Antibody-drug conjugates, their preparation and their therapeutic use

ABSTRACT

The present disclosure relates to conjugates comprising biologically active molecules linked to a multimeric antigen-binding compound or a multimeric immunoglobulin via a linker. The disclosure further provides reagents and methods of manufacturing the conjugates and the linkers. The disclosure also provides compositions comprising the conjugates, methods of modifying abnormal cell growth and methods of treatment using the conjugates or the compositions.

TECHNICAL FIELD

The disclosure relates to conjugates comprising biologically activemolecules connected to a multimeric antigen-binding compound through alinker compound. The disclosure also provides reagents and methods forconjugating the biologically active molecules to the multimericantigen-binding compound.

BACKGROUND

Antibody drug conjugate (ADC) are antibodies that are conjugated, viachemical linkers, to cytotoxic agents. ADCs leverage an antibody'sbinding specificity for its target to deliver cytotoxic agents to anabnormal cell. Traditional ADC technology involves chemically attachinga drug, through a linker, to particular amino acid residues of theantibody. For example, the linker can be attached to the antibody at thesulfhydryl groups of one or more cysteine residues within the antibodyheavy and/or light chains. A recognized problem with conjugation atcysteine residues, however, is that the process requires disrupting oneor more interchain disulfide bonds which ordinarily serve to maintainthe structure and function of the antibody molecule. Cysteine-conjugatedADCs can have reduced stability, which potentially impedes the bindingand therapeutic properties of the ADC. Thus, there is a need fortechniques for conjugating an antibody at its cysteine residues that donot significantly impact the antibody's binding affinity, e.g., methodsof conjugation that reconnect cysteine residues of an interchaindisulfide bond cleaved during the conjugation process.

SUMMARY

Provided herein are chemical linkers that connect a biologically activecompound, e.g., cytotoxic agent, to cysteine residues of a multimericantigen-binding compound, e.g., antibody, as well as conjugates thereof,pharmaceutical compositions comprising the same, methods of preparingthe same, and methods of treating disorders comprising administering thesame.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a multimeric immunoglobulin conjugate.

FIG. 2 shows a SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gelelectrophoresis) of an IgG1 Antibody-bromomethylacrylate (1) conjugateunder reducing and non-reducing conditions.

FIG. 3 shows a SDS-PAGE of an IgG1 antibody conjugated to exemplarysmall molecule conjugates 1, 2, 6, and 9 under reducing and non-reducingconditions.

FIG. 4 shows a SDS-PAGE of an anti-PRLR antibody conjugated to exemplarylinker payloads H1H6765P-26, H1H6765P-28 and H1H6765P-29 under reducingand non-reducing conditions.

FIG. 5 shows an ESI MS (electro spray ionization mass spectrometry)spectrum of the conjugate H1H6765P-28.

FIG. 6 shows an ESI MS spectrum of the conjugate H1H6765P-26.

FIG. 7 shows the selective anti-proliferation ability of conjugateH1H6765P-26 in HEK293, HEK293/hPRLR, and T47D.

FIG. 8 shows the selective anti-proliferation ability of conjugateH1H6765P-28 and H1H6765P-29 in HEK293, HEK293/hPRLR, and T47D.

DETAILED DESCRIPTION

The references to certain embodiments made in the following descriptionare considered illustrative only of the principles of the disclosure.Further, since numerous modifications and changes will readily beapparent to those skilled in the art, it is not intended to limit thedisclosure to the exact construction and process shown as describedherein. Accordingly, all suitable modifications and equivalents may beresorted to as falling within the scope of the disclosure and as definedby the claims that follow.

I. DEFINITIONS

The words “comprise”, “comprising”, “include” and “including” when usedin this specification and in the following claims are intended tospecify the presence of the stated features, integers, components, orsteps, but they do not preclude the presence or addition of one or moreadditional features, integers, components, or steps thereof.

General terms used in any of the embodiments herein can be defined asfollows; however, the meaning stated should not be interpreted aslimiting the scope of the term per se.

The symbol

denotes the points of attachment.

The term “conjugate” as used herein refers to compound having amultimeric antigen-binding compound or a multimeric immunoglobulin, alinker and a biologically active molecule. Illustrative examples includecompounds of formula (I), and FIG. 1).

The term “spacer” as used herein refers to chemical building blocks ofthe linker used to spatially separate the multimeric antigen-bindingcompound or a multimeric immunoglobulin from the biologically activemolecule and to allow for catabolism of the linker inside of cells. Aspacer can be represented by Z₁ and Z₂.

The term “alkyl” as used herein refers to a hydrocarbon group having ageneral formula C_(n)H_(2n+1). Examples of alkyl include, but are notlimited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, and the like.Further exemplary alkyl groups include, but are not limited to, thosethat have from one to ten carbon atoms, one to nine carbon atoms, one toeight carbon atoms, one to seven carbon atoms, one to six carbon atoms,one to five carbon atoms, one to four carbon atoms, one to three carbonatoms, one to two carbon atoms or one carbon atom.

The term “aryl” as used herein refers to a monovalent or polycyclicaromatic hydrocarbon. Exemplary aryl groups include, but are not limitedto, those having 6 to 18 carbon atoms. Exemplary aryl groups furtherinclude, but are not limited to, phenyl, substituted phenyl,naphthalenyl, anthracenyl, indenyl, tetrahydronapthyl and the like.

The term “alkenyl” as used herein refers to an aliphatic linear orbranched monovalent hydrocarbon radical of two or more carbon atoms withat least one site of unsaturation. Examples of alkenyl groups include,but are not limited to, ethylenyl, vinyl, allyl, and the like.

The term “alkynyl” as used herein refers to a univalent aliphatichydrocarbon radical containing at least one triple bond. Exemplaryalkynyl groups include, but are not limited to, those having from two totwenty carbon atoms (and include at least one triple bond). Exemplaryalkynyl groups also include, but are not limited to, ethynyl, propynyl,1-butynyl, 2-butynyl, 1-pentynyl, hexynyl and the like.

The term “cycloalkyl” as used herein, refers to a monovalent saturatedcarbocyclic ring radical. Exemplary cycloalkyl groups include thosehaving 3 to 7 ring carbon atoms. Exemplary cycloalkyl groups include,but are not limited to, cyclopropyl, cyclopentyl, and cyclohexyl.

The term “heteroaryl” as used herein, refers to a monovalent aromaticradical containing at least one heteroatom in its aromatic ring.Heteroaryl groups include, but are not limited to fused ring systems (atleast one must be aromatic) that include up to 5 to 18 atoms, containingone or more heteroatoms independently selected from nitrogen, sulfur oroxygen. Illustrative heteroaryl groups further include, but are notlimited to, pyridinyl, triazolyl, furyl, pyrazinyl, thienyl, isoxazolyl,indazolyl, furazanyl, benzothiazolyl, quinazolinyl, and furopyridinyl.

The term “heterocyclyl” as used herein refers to saturated or partiallysaturated carbocyclic radical, including, but not limited to thosehaving 3 to 18 carbon atoms, in which at least one ring atom is aheteroatom selected from nitrogen, oxygen, phosphorous, and sulfur. Aheterocycyl can be a monocycle or a bicycle, for example. Exemplaryheterocyclyl groups include, but are not limited to, pyrolidinyl,tetrahydrofuranyl, dihydropyranyl, thioxanyl, 2H-pyranyl, dioxanyl,dithianyl, piperidino, and the like.

The phrase “pharmaceutically acceptable salt” as used herein refers toboth organic and inorganic salts of the conjugate compounds describedherein, e.g., compounds of formula (I), FIG. 1), formula (III). Thesalts are pharmaceutically acceptable and include: sulfate, citrate,nitrate, phosphate, ascorbate, chloride, bromide, gluconate, benzoate,oxalate, pantothenate, and the like. Note that pharmaceuticallyacceptable salts herein may include more than one charged atom in itsstructure as well as one or more counter ion. Preparation of conjugatecompounds herein as pharmaceutically acceptable salts is well known toone of skill in the art.

The term “therapeutically effective amount” as used herein refers to anamount that produces the desired effect for which it is administered.The exact amount will depend on the purpose of the treatment, and willbe ascertainable by one skilled in the art using known techniques (see,for example, Lloyd (1999) The Art, Science and Technology ofPharmaceutical Compounding).

II. MULTIMERIC ANTIGEN-BINDING COMPOUND CONJUGATES

The present disclosure provides multimeric antigen-binding compoundconjugates comprising the formula (I):

wherein: IC1 is a first immunoglobulin chain, and IC2 is a secondimmunoglobulin chain; further wherein IC1 and/or IC2, alone or togethercomprise at least one antigen-binding domain; Z₁ and Z₂ are eachindependently absent or a spacer; E is O, S, NR₄, or CR₅R₆; D is abiologically active molecule; A is absent, a natural or non-naturalamino acid, or a peptide comprising 2-20 amino acids; W is absent, —O—,—S—, —CR₅R₆—, or —NR₄—; X is absent, aryl, heteroaryl, cycloalkyl, orheterocyclyl, wherein aryl, heteroaryl, cycloalkyl, and heterocyclyl areoptionally substituted; and Y is absent,

wherein: A₁, A₃, R₁ and R₃ are each independently absent, an amino acid,a peptide having 2-20 amino acids, an alkyl, an alkynyl, an alkenyl, acycloalkyl, an aryl, a heteroaryl, a heterocyclyl, —CR₅R₆—, —O—,—C(═O)—, —O—C(═O)—, —C(═O)—O—, —O—C(═O)—O—, —C(═O)-(CH_(x))_(p1)—,—C(═O)—O—(CH_(x))_(p1)—, —(CH_(x))_(p1)—C(═O)—, —(CH_(x))_(p1)-C(═O)—O—,—(O—(CH₂)_(p2)—)_(p3)—, —((CH₂)_(p2)-O—)_(p3)—, —C(═S)—, —C(═S)—S—,—S—C(═S)—, —C(═S)—NH—, —S—C(═S)—S—, —S—, —SO—, —SO₂—, —NR₄—,—N(R₄)—C(═O)—N(R₈)—, —N(R₄)—C(═O)O—, —N(R₄)—C(═O)—, —C(═O)—N(R₄)—,—C(═O)—N(R₄)—C(═O)—, or —O—C(═O)—NR₄—,

wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, andheterocyclyl are optionally substituted; A₄ and A₅ are eachindependently —O—, —S—, —NR₁₈—, or —CR₅R₆—; R₁₇ is O, S, NR₁₈, or CR₅R₆;R₁₈ is H, alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl,heterocyclyl, or acyl,

wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl,heterocyclyl, and acyl are optionally substituted; R₄, R₅, R₆ and R₈ areeach independently H or a substituted or unsubstituted: alkyl, alkenyl,alkynyl, aryl, heteroaryl, or heterocyclyl; p1, p2 and p3 are eachindependently 0, or an integer from 1 to 100; and x is 0, 1 or 2.

In one embodiment, multimeric immunoglobulin or antigen-bindingcompounds are any molecules capable of binding with some specificity toa given binding partner under physiological conditions. In some aspectsthe multimeric immunoglobulin or antigen-binding compound is capable ofbinding to a cell or cell population.

In an embodiment, the disclosure provides the antigen-bindingcompound-conjugate represented by formula (I), wherein IC1 is a firstheavy chain of an antibody or antigen-binding portion thereof; and IC2is a second heavy chain of the antibody or antigen-binding portionthereof. In certain sub-embodiments wherein IC1 and IC2 are antibodyheavy chains, one or more of IC1 and/or IC2 may each be independentlyassociated with or connected to an antibody light chain orantigen-binding portion thereof (e.g., forming a complete tetramericantibody structure). In a further embodiment, the disclosure providesthe antigen-binding compound-conjugate represented by formula (I),wherein the antigen-binding compound-conjugate is substantially stableunder reducing conditions. In a further embodiment, the disclosureprovides the antigen-binding compound-conjugate represented by formula(I), wherein D is a cytotoxic agent.

In an embodiment, the disclosure provides the antigen-bindingcompound-conjugate represented by formula (I), wherein IC1 is a heavychain of an antibody or antigen-binding portion thereof; and IC2 is alight chain of the antibody or antigen-binding portion thereof. Incertain sub-embodiments wherein IC1 is an antibody heavy chain and IC2is an antibody light chain, the IC1/IC2 construct may be associated withor connected to another antibody heavy chain/light chain pair (e.g.,forming a complete tetrameric antibody structure). In a furtherembodiment, the disclosure provides the antigen-bindingcompound-conjugate represented by formula (I), wherein theantigen-binding compound-conjugate is substantially stable underreducing conditions. In a further embodiment, the disclosure providesthe antigen-binding compound-conjugate represented by formula (I),wherein D is a cytotoxic agent. In a further embodiment, the disclosureprovides the antigen-binding compound-conjugate represented by formula(I), wherein the antigen-binding compound-conjugate specifically binds atumor-associated antigen.

In an embodiment, the disclosure provides the antigen-bindingcompound-conjugate represented by formula (I), wherein theantigen-binding compound-conjugate is substantially stable underreducing conditions and/or in the presence of one or more reducingagents. Reducing agents, for the purpose of the present disclosure,include any agent that can rupture or disrupt a disulfide bond. Examplesof suitable reducing agents include dithiothreitol (DTT),2-mercaptoethanol (BME), 2-mercaptoethylamine (MEA), sodiummethanethiolate, sodium 2-sulfanylethanesulfonate, cysteine, tris(2-carboxyethyl) phosphine (TCEP), and derivatives of any of theforegoing. Reducing conditions include incubation in the presence of, ortreatment with, one or more reducing agents and/or high temperature. Ina further embodiment, the disclosure provides the antigen-bindingcompound-conjugate represented by formula (I), wherein D is a cytotoxicagent. In a further embodiment, the disclosure provides theantigen-binding compound-conjugate represented by formula (I), whereinthe antigen-binding compound-conjugate specifically binds atumor-associated antigen.

In an embodiment, the disclosure provides the antigen-bindingcompound-conjugate represented by formula (I), wherein D is a cytotoxicagent (also referred to herein as “biologically active molecules”). Asused herein, cytotoxic agents include any agent that is detrimental tothe growth, viability or propagation of cells. Examples of cytotoxicagents that can be used in the context of the present disclosureinclude, e.g., 1-(2chloroethyl)-1,2-dimethanesulfonyl hydrazide,1,8-dihydroxy-bicyclo[7.3.1]trideca-4,9-diene-2,6-diyne-13-one,1-dehydrotestosterone, 5-fluorouracil, 6-mercaptopurine, 6-thioguanine,9-amino camptothecin, actinomycin D, amanitins, aminopterin, anguidine,anthracycline, anthramycin (AMC), auristatins, bleomycin, busulfan,butyric acid, calicheamicins, camptothecin, carminomycins, carmustine,cemadotins, cisplatin, colchicin, combretastatins, cyclophosphamide,cytarabine, cytochalasin B, dactinomycin, daunorubicin, decarbazine,diacetoxypentyldoxorubicin, dibromomannitol, dihydroxy anthracin dione,disorazoles, dolastatin, doxorubicin, duocarmycin, echinomycins,eleutherobins, emetine, epothilones, esperamicin, estramustines,ethidium bromide, etoposide, fluorouracils, geldanamycins, gramicidin D,glucocorticoids, irinotecans, leptomycins, leurosines, lidocaine,lomustine (CCNU), maytansinoids, mechlorethamine, melphalan,mercatopurines, methopterins, methotrexate, mithramycin, mitomycin,mitoxantrone, N8-acetyl spermidine, podophyllotoxins, procaine,propranolol, pteridines, puromycin, pyrrolobenzodiazepines (PBDs),rhizoxins, streptozotocin, tallysomycins, taxol, tenoposide, tetracaine,thioepa chlorambucil, tomaymycins, topotecans, tubulysin, vinblastine,vincristine, vindesine, vinorelbines, spliceostatin, amanatin, orcalicheamicin and derivatives of any of the foregoing. According tocertain embodiments, the cytotoxic agent is a maytansinoid such as DM1or DM4, a tomaymycin derivative, or a dolastatin derivative. Othercytotoxic agents known in the art are contemplated within the scope ofthe present disclosure, including, e.g., protein toxins such ricin, C.difficile toxin, pseudomonas exotoxin, ricin, diphtheria toxin,botulinum toxin, bryodin, saporin, pokeweed toxins (i.e.,phytolaccatoxin and phytolaccigenin), and others such as those set forthin Sapra et al., Pharmacol. & Therapeutics, 2013, 138:452-469.

In an embodiment, the disclosure provides the antigen-bindingcompound-conjugate represented by formula (I), wherein theantigen-binding compound-conjugate specifically binds a tumor-associatedantigen.

In one embodiment, the disclosure provides antigen-bindingcompound-conjugate represented by formula (I), wherein Z₂ is representedby the following structural formula:

—Z_(2A)-Z_(2B)-Z_(2C)-Z_(2D)—,

wherein: Z_(2A), Z_(2B), Z_(2C)and Z_(2D) are each independently absent,an amino acid, a peptide having 2-20 amino acids, an alkyl, an alkynyl,an alkenyl, a cycloalkyl, an aryl, a heteroaryl, a heterocyclyl,—CR₅R₆—, —O—, —C(═O)—, —O—C(═O)—, —C(═O)—O—, —O—C(═O)—O—,—C(═O)—(CH_(x))_(p1), —C(═O)—O—(CH_(x))_(p1), —(CH_(x))_(p1)—C(═O)—,—(CH_(x)) —C(═O)—O—, —(O—CH₂)_(p2)—)_(p3)—, —((CH₂)_(p2)—O—)_(p3)—,—C(═S)—, —C(═S)—S—, —C(═S)—NH—, —S—C(═S)—, —S—C(═S)—S—, —S—, —SO—, SO₂—,—NR₄—, —N(R₄)—C(═O)-N(R₈)—, —N(R₄)—C(═O)O—, —N(R₄)—C(═O)—,—C(═O)—N(R₄)—, —C(═O)—N(R₄)—C(═O)—, —O—C(═O)—N(R₄), —O—C(═S)—N(R₄)—, or—C(═S)—N(R₄)—, wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl,heteroaryl, and heterocyclyl are optionally substituted and R₄, R₅, R₆and R₈ are each independently H or a substituted or unsubstituted:alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl; and Z₁ isrepresented by the following structural formula:

—Z_(1A)-Z_(1B)-Z_(1C)-Z_(1D)—,

wherein: Z_(1A), Z_(1B), Z_(1C) and Z_(1D) are each independentlyabsent, an amino acid, a peptide having 2-20 amino acids, an alkyl, analkynyl, an alkenyl, a cycloalkyl, an aryl, a heteroaryl, aheterocyclyl, —CR₅R₆—, —O—, —C(═O)—, —O—C(═O)—, —C(═O)—O—, —O—C(═O)—O—,—C(═O)—(CH_(x))_(p1), —C(═O)—O—(CH_(x))_(p1),—CH_(x))_(p1)—C(═O)—,—(CH_(x))_(p1)—C(═O)—O—, —(O—(CH₂)_(p2)—)_(p3)—,—(CH₂)_(p2)—O—)_(p3)—, —C(═S)—, —C(═S)—S—, —C(═S)—NH—, —S—C(═S)—,—S—C(═S)—S—, —S—, —SO—, —SO₂-, —NR₄-, —N(R₄)—C(═O)—N(R₈)—,—N(R₄)—C(═O)O—, —N(R₄)—C(═O)—, —C(═O)—N(R₄)—, C(═O)—N(R₄)—C(═O)—,—O—C(═O)—N(R₄), —O—C(═S)—N(R₄)—, or —C(═S)—N(R₄)—,

wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, andheterocyclyl are optionally substituted and R₄, R₅, R₆ and R₈ are eachindependently H or a substituted or unsubstituted: alkyl, alkenyl,alkynyl, aryl, heteroaryl, or heterocyclyl; p1, p2 and p3 are eachindependently 0, or an integer from 1 to 100; and x is 0,1 or 2.

The disclosure also provides a multimeric immunoglobulin conjugatehaving the structure as set forth in FIG. 1):

wherein one or more M1, M2, M3, and/or M4 are each independently absent(i.e., the adjacent S atoms are directly connected to one another via adisulfide bond), or have the structure represented by formula (II)

wherein LC1 is a first antibody light chain, LC2 is a second antibodylight chain, HC1 is a first antibody heavy chain, and HC2 is a secondantibody heavy chain;

wherein LC1, LC2, HC1 and/or HC2 comprise at least one antigen-bindingdomain; E is O, S, NR₄, or CR₅R₆; D is a biologically active molecule; Ais absent, a natural or non-natural amino acid, or a peptide comprising2-20 amino acids; W is absent, —O—, —S—, —CR₅R₆—, or —NR₄—; X is absent,aryl, heteroaryl, cycloalkyl, heterocyclyl, wherein aryl, heteroaryl,cycloalkyl, and heterocyclyl are optionally substituted; and Y isabsent,

wherein A₁, A₃, R₁ and R₃ are each independently absent, an amino acid,a peptide having 2-20 amino acids, an alkyl, an alkynyl, an alkenyl, acycloalkyl, an aryl, a heteroaryl, a heterocyclyl, —CR₅R₆—, —O—,—C(═O)—, —O—C(═O)—, —C(═O—)—O—, —O—-C(═O)—O—, —C(═O)—(CH_(x))_(p1)—,—C(═O)—O—(CH_(x))_(p1)—, —(CH_(x))_(p1)—C(═O)—, —(CH_(x))_(p1)—C(═O)—O—,—O—(CH₂)_(p2))_(p3), ((CH₂)_(p2)—O—)_(p3)—, —C(═S)—, —C(═S)—S—,—S—C(═S)—, —C(═S)—NH—, —S—C(═S)—S—, —S—, —SO—, —SO₂—, —NR₄—,—N(R₄)—C(═O)—N(R₈)—, —N(R₄)—C(═O)O—, —N(R₄)—C(═O)—, —C(═O)—N(R₄)—,—C(═O)—N(R₄)—C(═O)—, or —O—C(═O)—NR₄—, wherein alkyl, alkynyl, alkenyl,cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionallysubstituted; A₄ and A₅ are each independently —O—, —S—, —NR₁₈—, or—CR₅R₆—; R₁₇ is O, S, NR₁₈, or CR₅R₆; R₁₈ is H, alkyl, alkynyl, alkenyl,cycloalkyl, aryl, heteroaryl, heterocyclyl, or acyl, wherein alkyl,alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, and acylare optionally substituted; R₄, R₅, R₆ and R₈ are each independently Hor a substituted or unsubstituted: alkyl, alkenyl, alkynyl, aryl,heteroaryl, or heterocyclyl; p1, p2 and p3 are each independently 0, oran integer from 1 to 100; and x is 0, 1 or 2.

In an embodiment, the disclosure provides a multimeric immunoglobulinconjugate having the structure as set forth in FIG. 1), wherein Z₂ isrepresented by the following structural formula:

—Z_(2A)-Z_(2B)-Z_(2C)-Z_(2D)—,

wherein: Z_(2A), Z_(2B), Z_(2C) and Z_(2D) are each independentlyabsent, an amino acid, a peptide having 2-20 amino acids, an alkyl, analkynyl, an alkenyl, a cycloalkyl, an aryl, a heteroaryl, aheterocyclyl, —CR₅R₆—, —O—, —C(═O)—, —O—C(═O)—, —C(═O)—O—, —O—C(═O)—O—,—C(═O)—(CH_(x))_(p1)—, —C(═O)—O—(CH_(x))_(p1)—, —(CH_(x))_(p1)—C(═O)—,—(CH_(x))_(p1)—C(═O)—O—, —(O—(CH₂)_(p2)—)_(p3)—, —((CH₂)_(p2)—O—)_(p3)—, —C(═S)—, —C(═S)—S—, —C(═S)—NH—, —S—C(═S)—, —S—C(═S)—S—,—S—, —SO—, —SO₂—, NR₄—, —N(R₄)—C(═O)—N(R₈)—, —N(R₄)—C(═O)O—,—N(R₄)—C(═O)—, —C(═O)—N(R₄)—, —C(═O)—N(R₄)—C(═O)—, —O—C(═O)—N(R₄),—O—C(═S)—N(R₄)—, or —C(═S)—N(R₄)—, wherein alkyl, alkynyl, alkenyl,cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionallysubstituted and R₄, R₅, R₆ and R₈ are each independently H or asubstituted or unsubstituted: alkyl, alkenyl, alkynyl, aryl, heteroaryl,or heterocyclyl; and Z₁ is represented by the following structuralformula:

—Z_(1A)-Z_(1B)-Z_(1C)-Z_(1D)—,

wherein: Z_(1A), Z_(1B), Z_(1C) and Z_(1D) are each independentlyabsent, an amino acid, a peptide having 2-20 amino acids, an alkyl, analkynyl, an alkenyl, a cycloalkyl, an aryl, a heteroaryl, aheterocyclyl, —CR₅R₆—, —O—, —C(═O)—, —O—C(═O)—, —C(═O)—O—, —O—C(═O)—O—,—C(═O)—(CH_(p1)—, —C(═O)—O—(CH_(x))_(p1)—, —(CH_(x))_(p1)—C(═O)—,—(CH_(x))_(p1)—C(═O)—O—, —(O—(CH₂)_(p2)—)_(p3), —((CH₂)_(p2)—O—)_(p3)—,—C(═S)—, —C(═S)—S—, —C(═S)—NH—, —S—C(═S)—, —S—C(═S)—S—, —S—, —SO—,—SO₂—, NR₄—, —N(R₄)—C(═O)—N(R₈)—, —N(R₄)—C(═O)O—, —N(R₄)—C(═O)—,—C(═O)—N(R₄)—, C(═O)—N(R₄)—C(═O)—, —O—C(═O)—N(R₄), —O—C(═S)—N(R₄)—, or—C(═S)—N(R₄)—, wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl,heteroaryl, and heterocyclyl are optionally substituted and R₄, R₅, R₆and R₈ are each independently H or a substituted or unsubstituted:alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl; p1, p2 andp3 are each independently 0, or an integer from 1 to 100; and x is 0, 1or 2.

In an embodiment, the disclosure provides a multimeric immunoglobulinconjugate having the structure as set forth in FIG. 1) wherein theconjugate is substantially stable under reducing conditions. In afurther embodiment, the disclosure provides a multimeric immunoglobulinconjugate having the structure as set forth in FIG. 1) wherein theconjugate specifically binds a tumor-associated antigen.

In an embodiment, the disclosure provides a multimeric immunoglobulinconjugate having the structure as set forth in FIG. 1) wherein theconjugate specifically binds a tumor-associated antigen.

A. Antibodies and Multimeric Antigen-Binding Compounds

In certain embodiments, a multimeric immunoglobulin or anantigen-binding compound for use herein include antibodies. The term“antibody”, as used herein, means any antigen-binding molecule ormolecular complex comprising at least one complementarity determiningregion (CDR) that specifically binds to or interacts with a particularantigen. The term “antibody” includes immunoglobulin moleculescomprising four polypeptide chains, two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds, as well as multimersthereof (e.g., IgM). Each heavy chain comprises a heavy chain variableregion (abbreviated herein as HCVR or V_(H)) and a heavy chain constantregion. The heavy chain constant region comprises three domains, C_(H)1, C_(H) 2 and C_(H) 3. Each light chain comprises a light chainvariable region (abbreviated herein as LCVR or V_(L)) and a light chainconstant region. The light chain constant region comprises one domain(C_(L) 1). The V_(H) and V_(L) regions can be further subdivided intoregions of hypervariability, termed complementarity determining regions(CDRs), interspersed with regions that are more conserved, termedframework regions (FR). Each V_(H) and V_(L) is composed of three CDRsand four FRs, arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In differentembodiments of the disclosure, the FRs of an antibody may be identicalto the human germline sequences, or may be naturally or artificiallymodified. An amino acid consensus sequence may be defined based on aside-by-side analysis of two or more CDRs.

The term “antibody”, as used herein, also includes antigen-bindingfragments of full antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3-CDR3-FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDRwhich is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present disclosure include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (v)V_(H)-C_(H)-C_(H)2-C_(H)3; (vi) V_(H)-C_(H)2-C_(H)3; (vii) V_(H)-C_(L);(viii) V_(L)-C_(H)1; (ix) V_(L)-C_(H)2; (x) V_(L)-C_(H)3; (xi)V_(L)-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present disclosure maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may bemonospecific or multispecific (e.g., bispecific). A multispecificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multispecific antibody format may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe present disclosure using routine techniques available in the art.

In certain embodiments of the disclosure, the antibodies of thedisclosure are human antibodies. The term “human antibody”, as usedherein, is intended to include antibodies having variable and constantregions derived from human germline immunoglobulin sequences. The humanantibodies of the disclosure may include amino acid residues not encodedby human germline immunoglobulin sequences (e.g., mutations introducedby random or site-specific mutagenesis in vitro or by somatic mutationin vivo), for example in the CDRs and in particular CDR3. However, theterm “human antibody”, as used herein, is not intended to includeantibodies in which CDR sequences derived from the germline of anothermammalian species, such as a mouse, have been grafted onto humanframework sequences.

The antibodies of the disclosure may, in some embodiments, berecombinant human antibodies. The term “recombinant human antibody”, asused herein, is intended to include all human antibodies that areprepared, expressed, created or isolated by recombinant means, such asantibodies expressed using a recombinant expression vector transfectedinto a host cell (described further below), antibodies isolated from arecombinant, combinatorial human antibody library (described furtherbelow), antibodies isolated from an animal (e.g., a mouse) that istransgenic for human immunoglobulin genes (see e.g., Taylor et al.(1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed,created or isolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies have variable and constant regions derived from humangermline immunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the V_(H) andV_(L) regions of the recombinant antibodies are sequences that, whilederived from and related to human germline V_(H) and V_(L) sequences,may not naturally exist within the human antibody germline repertoire invivo.

In embodiments in which the multimeric immunoglobulin or antigen-bindingcompound for use herein is an antibody or antigen-binding fragmentthereof, the antibody or antigen-binding fragment binds to one or moreantigen binding partner. The antigen-binding partner may be apolypeptide such as a transmembrane molecule (e.g., receptor) or agrowth factor. Exemplary antigens include, but are not limited to,molecules such as renin; a growth hormone, including human growthhormone and bovine growth hormone; growth hormone releasing factor;parathyroid hormone; thyroid stimulating hormone; lipoproteins;alphal-antitrypsin; insulin A-chain; insulin B-chain; proinsulin;follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon;clotting factors such as factor vmc, factor IX, tissue factor (TF), andvon Willebrands factor; anti-clotting factors such as Protein C; atrialnatriuretic factor; lung surfactant; a plasminogen activator, such asurokinase or human urine or tissue-type plasminogen activator (t-PA);bombesin; thrombin; hemopoietic growth factor; tumor necrosisfactor-alpha and -beta; enkephalinase; RANTES (regulated on activationnormally T-cell expressed and secreted); human macrophage inflammatoryprotein (M1P-I-alpha); a serum albumin, such as human serum albumin;Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;prorelaxin; mouse gonadotropin-associated peptide; a microbial protein,such as betalactamase; DNase; 19E; a cytotoxic T-lymphocyte associatedantigen (CTLA), such as CTLA-4; inhibin; activin; vascular endothelialgrowth factor (VEGF); receptors for hormones or growth factors; proteinA or D; rheumatoid factors; a neurotrophic factor such as bone-derivedneurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT4,NT-5, or NT-6), or a nerve growth factor such as NGF-f3;platelet-derived growth factor (PDGF); fibroblast growth factor such asaFGF and bFGF; fibroblast growth factor receptor 2 (FGFR2), epidermalgrowth factor (EGF); transforming growth factor (TGF) such as TGF-alphaand TGF-beta, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5;insulin-like growth factor-1 and -II (IGF-1 and IGF-II); des(I-3)-IGF-1(brain IGF-1), insulin-like growth factor binding proteins, EpCAM, GD3,FLT3, PSMA, PSCA, MUCI, MUCI6, STEAP, CEA, TENB2, EphA receptors, EphBreceptors, folate receptor, FOLRI, mesothelin, cripto, alphavbeta6,integrins, VEGF, VEGFR, EGFR, transferrin receptor,1RTAI,1RTA2,1RTA3,1RTA4,1RTA5; CD proteins such as CD2, CD3, CD4, CDS,CD6, CD8, CDII, CDI4, CDI9, CD20, CD21, CD22, CD25, CD26, CD28, CD30,CD33, CD36, CD37, CD38, CD40, CD44, CD52, CD55, CD56, CD59, CD70, CDI9,CD80. CD81, CD103, CD105, CD134, CD137, CD138, CDI52, or an antibodywhich binds to one or more tumor-associated antigens or cell-surfacereceptors disclosed in US Publication No. 2008/0171040 or US PublicationNo. 2008/0305044 and incorporated in their entirety by reference;erythropoietin; osteoinductive factors; immunotoxins; a bonemorphogenetic protein (BMP); an interferon, such as interferon-alpha,-beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF,GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superoxidedismutase; T-cell receptors; surface membrane proteins; decayaccelerating factor; viral antigen such as, for example, a portion ofthe HIV envelope; transport proteins; homing receptors; addressins;regulatory proteins; integrins, such as CD11a, CD11b, CDllc, CDI8, anICAM, VLA-4 and VCAM; a tumor associated antigen such as AFP, ALK, B7H4,BAGE proteins, β-catenin, brc-abl, BRCA1, BORIS, CA9 (carbonic anhydraseIX), caspase-8, CD20, CD40, CD123, CDK4, CEA, CLEC12A, c-kit, cMET,CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, endoglin, Epcam, EphA2,ErbB2/Her2, ErbB3/Her3, ErbB4/Her4, ETV6-AML, Fra-1, FOLR1, GAGEproteins (e.g., GAGE-1, -2), GD2, GD3, GloboH, glypican-3, GM3, gp100,Her2, HLA/B-raf, HLA/EBNA1, HLA/k-ras, HLA/MAGE-A3, hTERT, IGF1R, LGR5,LMP2, MAGE proteins (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1,mesothelin, ML-IAP, Muc1, Muc16 (CA-125), MUM1, NA17, NGEP, NY-BR1,NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1,PDGFR-a, PDGFR-f3, PDGF-A, PDGF-B, PDGF-C, PDGF-D, PLAC1, PRLR, PRAME,PSCA, PSGR, PSMA (FOLH1), RAGE proteins, Ras, RGS5, Rho, SART-1, SART-3,Steap-1, Steap-2, STn, survivin, TAG-72, TGF-β, TMPRSS2, Tn, TNFRSF17,TRP-1, TRP-2, tyrosinase, and uroplakin-3, and fragments of any of theabove-listed polypeptides.

In some embodiments, the anti-body is an anti-PRLR antibody, e.g., thosedisclosed in U.S. Patent Publication No. 2015/0056222 A1.

Embodiments herein are target specific for therapeutic use. In oneembodiment, multimeric immunoglobulin or antigen-binding compound areprepared to interact with and bind to antigens defined as tumorantigens, which include antigens specific for a type of tumor orantigens that are shared, overexpressed or modified on a particular typeof tumor. Illustrative examples include: alpha-actinin-4 with lungcancer, ARTC1 with melanoma, BCR-ABL fusion protein with chronic myeloidleukemia, B-RAF, CLPP or Cdc27 with melanoma, CASP-8 with squamous cellcarcinoma, and hsp70-2 with renal cell carcinoma as well as thefollowing shared tumor-specific antigens, for example: BAGE-1, GAGE,GnTV, KK-LC-1, MAGE-A2, NA88-A, TRP2-INT2.

B. Biologically Active Molecules

Biologically active molecules herein (also referred to herein as“drugs,” “toxins,” “cytotoxic agents,” “chemotherapeutic agents,” andthe like) include any molecules that have a therapeutic use in a mammalwhen targeted to a specific cell, cell type, or tissue. In typicalembodiments the molecule is beneficially delivered to a target withinthe mammal and in particular is beneficially delivered to and thenwithin a cell (e.g., endocytosis) as compared to molecules released intothe vascular or lymphatic systems.

In one aspect, biologically active molecules are compounds that resultin the inhibition, retardation, reduction, and/or prevention of cellgrowth. Biologically active molecules can also result in cell death vianecrosis or apoptosis. Illustrative biologically active molecules foruse in conjugate compounds described herein include: maytansinoids(e.g., DM1, DM4, or derivative thereof, etc.), auristatins (e.g., MMAE,MMAD, MMAF, etc.), duocarmycin (e.g., MGBA), dolastatin, toxoids, andother chemotherapeutically effective drugs. In some embodiments,biologically active molecule (D) has the following structure:

Other specific examples of biologically active molecules that can beused in the context of the present disclosure include, e.g.,1-dehydrotestosterone, 2-pyrrolinodoxorubicin, 5-fluorouracil,6-mercaptopurine, 6-thioguanine, amanitin, actinomycin D, anthracycline(e.g., PNU-159682), anthramycin (AMC), bleomycin, busulfan,calicheamicins, carmustine cisplatin, colchicin,cyanomorpholino-doxorubicin, cyclophosphamide, cytarabine, cytochalasinB, dactinomycin, daunorubicin, decarbazine, dibromomannitol, dihydroxyanthracin dione, doxorubicin, emetine, epirubicin, ethidium bromide,etoposide, gramicidin D, glucocorticoids, lidocaine, lomustine (CCNU),mechlorethamine, melphalan, methotrexate, mithramycin, mitomycin,mitoxantrone, morpholino-doxorubicin, procaine, propranolol, puromycin,pyrrolobenzodiazapines, sibiromycin, streptozotocin, taxol, tenoposide,tetracaine, thioepa chlorambucil, trichothecenes, tubulysin,vincristine, and stereoisomers, isosteres, analogs or derivatives of anyof the foregoing.

In one embodiment the biologically active molecule is a maytansinoid ora maytansinoid analog. Exemplary maytansinoids for use herein aredescribed in Widdison et al., J. Med. Chem., 2006, 49, 4392-4408,incorporated by reference herein for all purposes. In some embodiments,the biologically active molecule is a maytansinoid described in WO2014/145090A1.

Examples of cytotoxic agents that can be used in the context of thepresent disclosure also include, but are not limited to,1-(2chloroethyl)-1,2-dimethanesulfonyl hydrazide,1,8-dihydroxy-bicyclo[7.3.1]trideca-4,9-diene-2,6-diyne-13-one,1-dehydrotestosterone, 5-fluorouracil, 6-mercaptopurine, 6-thioguanine,9-amino camptothecin, actinomycin D, amanitins, aminopterin, anguidine,anthracycline, anthramycin (AMC), auristatins, bleomycin, busulfan,butyric acid, calicheamicins, camptothecin, carminomycins, carmustine,cemadotins, cisplatin, colchicin, combretastatins, cyclophosphamide,cytarabine, cytochalasin B, dactinomycin, daunorubicin, decarbazine,diacetoxypentyldoxorubicin, dibromomannitol, dihydroxy anthracin dione,disorazoles, dolastatin, doxorubicin, duocarmycin, echinomycins,eleutherobins, emetine, epothilones, esperamicin, estramustines,ethidium bromide, etoposide, fluorouracils, geldanamycins, gramicidin D,glucocorticoids, irinotecans, leptomycins, leurosines, lidocaine,lomustine (CCNU), maytansinoids, mechlorethamine, melphalan,mercatopurines, methopterins, methotrexate, mithramycin, mitomycin,mitoxantrone, N8-acetyl spermidine, podophyllotoxins, procaine,propranolol, pteridines, puromycin, pyrrolobenzodiazepines (PBDs),rhizoxins, streptozotocin, tallysomycins, taxol, tenoposide, tetracaine,thioepa chlorambucil, tomaymycins, topotecans, tubulysin, vinblastine,vincristine, vinca alkyloids, vindesine, vinorelbines, spliceostatin,amanatin, or calicheamicin and derivatives of any of the foregoing.According to certain embodiments, the cytotoxic agent is a maytansinoidsuch as DM1 or DM4, a tomaymycin derivative, or a dolastatin derivative.Other cytotoxic agents known in the art are contemplated within thescope of the present disclosure, including, e.g., protein toxins suchricin, C. difficile toxin, pseudomonas exotoxin, ricin, diphtheriatoxin, botulinum toxin, bryodin, saporin, pokeweed toxins (i.e.,phytolaccatoxin and phytolaccigenin), and others such as those set forthin Sapra et al., Pharmacol. & Therapeutics, 2013, 138:452-469.

In an embodiment, the present disclosure relates to conjugates, e.g.,compounds of formula (I), formula (II), or formula (III), wherein thebiologically active molecule (D) is a cytotoxic biologically activemacrolide. In a further embodiment, the present disclosure providesmaytansinoid as represented by formula (I)(a) as biologically activemacrolide:

wherein A₆, A₇, A₈, A₉ are each independently absent, an amino acid,N-alkyl amino acid, a peptide having 2-20 amino acids, an alkyl, analkenyl, an alkynyl, a cycloalkyl, an aryl, a heteroaryl, aheterocyclyl, —CR₅R₆—, —O—, —C(═O)—, —O—, —C(═O)—, —C(═O)—O—,—O—C(═O)—O—, —C(═O)—(CH_(x))_(p1)—, —C(═O)—O—(CH_(x))_(p1),—(CH_(x))_(p1)—C(═O)—, —(CH_(x))_(p1)—C(═O)—O—, —(O—(CH₂)_(p2)—) _(p3)—,—((CH₂)_(p2)—O—)_(p3)—, —C(═S)—, —C(═S)—NH—, —C(═S)—S—, —S—C(═S)—,—S—C(═S)—S—, —S—, —SO—, —SO₂—, —NR₄—, —N(R₄)—C(═O)—N(R₈)—,—N(R₄)—C(═O)O—, —N(R₄)—C(═O)—, —C(═O)—N(R₄)—, —C(═O)—N(R₄)—C(═O)—,—O—C(═O)—NR₄, further wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl,heteroaryl, and heterocyclyl are optionally substituted; and R₄, R₅, R₆and R₈ are each independently H, or a substituted or unsubstituted:alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl; p1, p2 andp3 are each independently 0, or an integer from 1 to 100; and x is 0, 1or 2.

In some embodiments, the biologically active molecule (D) has thefollowing structure:

In a further embodiment, the maytansinoid is represented by thefollowing structural formula (II)(a):

C. Linkers

The linkers provided herein covalently connect a biologically activecompound (e.g., cytotoxic agent) to sulfhydryl groups (e.g., of cysteineresidues) of a multimeric antigen-binding compound (e.g., antibody). Incertain aspects, the linkers connect two sulfhydryl groups via 3-carbonbridge. Such linkers can serve to reconnect cysteine residues of anantibody disulfide that is cleaved during the conjugation process.

In an embodiment, the disclosure provides linker represented by formula(IV):

wherein: Z₂ and Z₁ are each independently absent or a spacer; A isabsent, a natural or non-natural amino acid, or a peptide comprising2-20 amino acids; W is absent, —O—, —S—, —CR₅R₆—, or —NR₄—; X is absent,aryl, heteroaryl, cycloalkyl, heterocyclyl, wherein aryl, heteroaryl,cycloalkyl, and heterocyclyl are optionally substituted;

Y is absent, wherein A_(l), A₃, R₁ and R₃ are each independently absent,an amino acid, a peptide having 2-20 amino acids, an alkyl, an alkynyl,an alkenyl, a cycloalkyl, an aryl, a heteroaryl, a heterocyclyl,—CR₅R₆—, —O—, —C(═O)—, —O—C(═O)—, —C(═O)—O—, —O—C(═O)—O—,—C(═O)—(CH_(x))_(p1)—, —C(═O)—O—CH_(x))_(p1)—, —(CH_(x))_(p1)—C(═O)—,—(CH_(x))_(p1)—C(═O)—O—, —(O—(CH₂)_(p2)—)_(p3)—, ((CH₂)_(p2)—O—)_(p3)—,—C(═S)—, —C(═S)—S—, —C(═S)—NH—, —S—C(═S)—, —S—C(═S)—S—, —S—, —SO—, —SO₂,—NR₄—, —N(R₄)—C(═O)—N(R₈)—, —N(R₄)—C(═O)O—, —N(R₄)—C(═O)—,—C(═O)—N(R₄)—, —C(═O)—N(R₄)—C(═O)—, or —O—C(═O)—NR₄—, wherein alkyl,alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl areoptionally substituted; A₄ and A₅ are each independently —O—, —S—,—NR₁₈—, or —CR₅R₆—; R₁₇ is O, S, NR₁₈, or CR₅R₆; R₁₈ is H, alkyl,alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, or acyl,wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl,heterocyclyl, and acyl are optionally substituted; R₄, R₅, R₆ and R₈ areeach independently H or a substituted or unsubstituted: alkyl, alkenyl,alkynyl, aryl, heteroaryl, or heterocyclyl; p1, p2 and p3 are eachindependently 0, or an integer from 1 to 100; and x is 0, 1 or 2.

In a further embodiment, the disclosure provides linker represented byformula (IV) wherein Z₂ is represented by the following structuralformula:

—Z_(2A)-Z_(2B)-Z_(2C)-Z_(2D)—,

wherein: Z_(2A), Z_(2B), Z_(2C) and Z_(2D) are each independentlyabsent, an amino acid, a peptide having 2-20 amino acids, an alkyl, analkynyl, an alkenyl, a cycloalkyl, an aryl, a heteroaryl, aheterocyclyl, —CR₅R₆—, —O—, —C(═O)—, —O—C(═O)—, —C(═O)—O—, —O—C(═O)—O—,—C(═O)—(CH_(x))_(p1)—, —C(═O)—O—(CH_(x))_(p1)—, (CH_(x))_(p1)—C(═O)—,—(CH_(x))_(p1)—C(═O)—O—, —(O—(CH₂)_(p2)—)_(p3)—, ((CH₂)_(p2)—O—)_(p3)—,—C(═S)—, —C(═S)—S—, —C(═S)—NH—, —S—C(═S)—, —S—C(═S)—S—, —S—, —SO—,—SO₂—, —NR₄—, —N(R₄)—C(═O)—N(R₈)—, —N(R₄)—C(═O)O—, —N(R₄)—C(═O)—,—C(═O)—N(R₄)—, —C(═O)—N(R₄)—C(═O)—, —O—C(═O)—N(R₄), —O—C(═S)—N(R₄)—, or—C(═S)—N(R₄)—, wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl,heteroaryl, and heterocyclyl are optionally substituted and R₄, R₅, R₆and R₈ are each independently H, or a substituted or unsubstituted:alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl; and whereinZ₁ is represented by the following structural formula:

—Z_(1A)-Z_(1B)-Z_(1C)-Z_(1D)—,

wherein: Z_(1A), Z_(1B), Z_(1C) and Z_(1D) are each independentlyabsent, an amino acid, a peptide having 2-20 amino acids, an alkyl, analkynyl, an alkenyl, a cycloalkyl, an aryl, a heteroaryl, aheterocyclyl, —CR₅R₆—, —O—, —C(═O)—, —O—C(═O)—, —C(═O)—O—, —O—C(═O)—O—,—C(═O)—(CH_(x))_(p1)—, —C(═O)—O—(CH_(x))_(p1)—, —(CH_(x))_(p1)—C(═O)—,—(CH_(x))_(p1)—C(═O)—O—, —(O—(CH₂)_(p2)—)_(p3)—,—((CH₂)_(p2))—0—)_(p3)—, —C(═S)—, —-C(═S)—S—, —C(═S)—NH—, —S—C(═S)—,—S—C(═S)—S—, —S—, —SO—, —SO₂—, NR₄—, —N(R₄)—C(═O)—N(R₈)—,—N(R₄)—C(═O)O—, —N(R₄)—C(═O)—, —C(═O)—N(R₄)—, C(═O)—N(R₄)—C(═O)—,—O—C(═O)—N(R₄), —O—C(═S)—N(R₄)—, or —C(═S)—N(R₄)—, wherein alkyl,alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl areoptionally substituted and R₄, R₅, R₆ and R₈ are each independently H,or a substituted or unsubstituted: alkyl, alkenyl, alkynyl, aryl,heteroaryl, or heterocyclyl; p1, p2 and p3 are each independently 0, oran integer from 1 to 100; and x is 0, 1 or 2.

In an embodiment, the maytansinoid provides part of the linker.

In an embodiment, the disclosure provides compounds of formula (I),formula (II), formula (III), or formula (IV), wherein A is an amino acidselected from the group consisting of alanine, aspartic acid, glutamicacid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine,methionine, asparagine, proline, glutamine, arginine, serine, threonine,valine, tryptophan, tyrosine, cysteine, and citrulline.

In an embodiment, the disclosure provides compounds of formula (I),formula (II), formula (III), or formula (IV), wherein A is a peptideselected from the group consisting of valine-citrulline,citrulline-valine, lysine-phenylalanine, phenylalanine-lysine,valine-asparagine, asparagine-valine, threonine-asparagine,serine-asparagine, asparagine-serine, phenylalanine-asparagine,asparagine-phenylalanine, leucine-asparagine, asparagine-leucine,isoleucine-asparagine, asparagine-isoleucine, glycine-asparagine,asparagine-glycine, glutamic acid-asparagine, asparagine-glutamic acid,citrulline-asparagine, asparagine-citrulline, alanine-asparagine,asparagine-alanine.

In an embodiment, the disclosure provides compounds of formula (I),formula (II), formula (III), or formula (IV), wherein X is an arylselected from the group consisting of

wherein R₉, R₁₀, R₁₁, and R₁₂ are each independently H, an alkyl,cycloalkyl, aryl, heteroaryl, heterocyclyl, halogen, NR₁₃R₁₄, nitro,cyano, —OH, —O—C(═O)—R₁₅, —C(═O)—R₁₅, —C(═O)—O—R₁₅, —C(═O)—NR₁₃ R₁₄; andfurther wherein, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclylare optionally substituted; and R₁₃ and R₁₄ are each independently H oran optionally substituted alkyl; and R₁₅ is an optionally substitutedalkyl.

In one aspect, the linkers are useful to covalently link ligands withtherapeutic agents and markers. In another aspect, the linkers improvechemical and/or systemic stability of the attached moieties. In anotheraspect, the linkers reduce in vivo toxicity of the attached moieties. Inanother aspect, the linkers improve pharmacokinetics, pharmacodynamics,and/or bioavailability of the attached moieties. In one aspect, thelinkers are cleavable linkers, i.e., the linkers can be cleaved andrelease a biologically active molecule at a site in or near a targetcell or a cell population in a pharmacologically effective form. Inanother aspect, the linkers are non-cleavable, but the antibody-drugconjugate can be degraded to release its attached moieties in apharmacologically effective form.

D. Exemplary Conjugates

In one aspect, the disclosure provides a multimeric antigen-bindingcompound-conjugate having the formula (I)

wherein: IC1 is a first immunoglobulin chain, and IC2 is a secondimmunoglobulin chain; further wherein IC1 and/or IC2 comprise at leastone antigen-binding domain; Z₁ and Z₂ are each independently absent or aspacer; E is O, S, NR₄, or CR₅R₆; D is a biologically active molecule; Ais absent or a natural or non-natural amino acid, or a peptidecomprising 2-20 amino acids; W is absent, —O—, —S—, —CR₅R₆—, —NR₄—; X isabsent, aryl, heteroaryl, cycloalkyl, heterocyclyl, wherein aryl,heteroaryl, cycloalkyl, and heterocyclyl are optionally substituted; andY is absent,

wherein A₁, A₃, R₁ and R₃ are each independently absent, an amino acid,a peptide having 2-20 amino acids, an alkyl, an alkynyl, an alkenyl, acycloalkyl, an aryl, a heteroaryl, a heterocyclyl, —CR₅R₆—, —O—,—C(═O)—, —O—C(═O)—, —C(═O)—O—, —O—C(═O)—O—, —C(═O)—(CH_(x))_(p1)—,—C(═O)—O—(CH_(x))_(p1)—, —(CH_(x))_(p1)—C(═O)—, —(CH_(x))_(p1)—C(═O)—O—,—(O—(CH₂)_(p2)—)_(p3)—, ((CH₂)_(p2)—O—)_(p3)—, —C(═S)—, —C(═S)—S—,—S—C(═S)—, —C(═S)—NH—, —S—C(═S)—S—, —S—, —SO—, —SO₂—, —NR₄—,—N(R₄)—C(═O)—N(R₈)—, —N(R₄)—C(═O)O—, —N(R₄)—C(═O)—, —C(═O)—N(R₄)—,—C(═O)—N(R₄)—C(═O)—, or —O—C(═O)—NR₄—, wherein alkyl, alkynyl, alkenyl,cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionallysubstituted; A₄ and A₅ are each independently —O—, —S—, —NR₁₈—, —CR₅R₆—;R₁₇ is O, S, NR₁₈, or CR₅R₆; R₁₈ is H, alkyl, alkynyl, alkenyl,cycloalkyl, aryl, heteroaryl, heterocyclyl, or acyl, wherein alkyl,alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, and acylare optionally substituted; R₄, R₅, R₆ and R₈ are each independently H,or a substituted or unsubstituted: alkyl, alkenyl, alkynyl, aryl,heteroaryl, or heterocyclyl; p1, p2 and p3 are each independently 0, oran integer from 1 to 100; and x is 0, 1 or 2.

In an embodiment, the disclosure provides the antigen-bindingcompound-conjugate represented by formula (I), wherein IC1 is a firstheavy chain of an antibody or antigen-binding portion thereof; and IC2is a second heavy chain of the antibody or antigen-binding portionthereof. In certain sub-embodiments wherein IC1 and IC2 are antibodyheavy chains, one or more of IC1 and/or IC2 may each be independentlyassociated with or connected to an antibody light chain orantigen-binding portion thereof (e.g., forming a complete tetramericantibody structure). In a further embodiment, the disclosure providesthe antigen-binding compound-conjugate represented by formula (I),wherein the antigen-binding compound-conjugate is substantially stableunder reducing conditions. In a further embodiment, the disclosureprovides the antigen-binding compound-conjugate represented by formula(I), wherein D is a cytotoxic agent.

In an embodiment, the disclosure provides the antigen-bindingcompound-conjugate represented by formula (I), wherein IC1 is a heavychain of an antibody or antigen-binding portion thereof; and IC2 is alight chain of the antibody or antigen-binding portion thereof. Incertain sub-embodiments wherein IC1 is an antibody heavy chain and IC2is an antibody light chain, the IC1/IC2 construct may be associated withor connected to another antibody heavy chain/light chain pair (e.g.,forming a complete tetrameric antibody structure). In a furtherembodiment, the disclosure provides the antigen-bindingcompound-conjugate represented by formula (I), wherein theantigen-binding compound-conjugate is substantially stable underreducing conditions. As used herein, the expression “substantiallystable,” when used in the context of an antibody or other multimericimmunoglobulin means that the re-joined disulfide linking two separatepolypeptide chains can hold the antibody or immunoglobulin substantiallyintact under reducing conditions such as the reducing environment of anSDS-PAGE gel, or in the presence of serum (e.g., human, monkey, bovine,mouse, rat, etc.) at 37° C., or at temperatures greater than 90° C.,etc. In a further embodiment, the disclosure provides theantigen-binding compound-conjugate represented by formula (I), wherein Dis a cytotoxic agent. In a further embodiment, the disclosure providesthe antigen-binding compound-conjugate represented by formula (I),wherein the antigen-binding compound-conjugate specifically binds atumor-associated antigen.

In an embodiment, the disclosure provides the antigen-bindingcompound-conjugate represented by formula (I), wherein theantigen-binding compound-conjugate is substantially stable underreducing conditions. In a further embodiment, the disclosure providesthe antigen-binding compound-conjugate represented by formula (I),wherein D is a cytotoxic agent. In a further embodiment, the disclosureprovides the antigen-binding compound-conjugate represented by formula(I), wherein the antigen-binding compound-conjugate specifically binds atumor-associated antigen.

In an embodiment, the disclosure provides the antigen-bindingcompound-conjugate represented by formula (I), wherein D is a cytotoxicagent, including, e.g., any cytotoxic agent as set forth elsewhereherein.

In an embodiment, the disclosure provides the antigen-bindingcompound-conjugate represented by formula (I), wherein theantigen-binding compound-conjugate specifically binds a tumor-associatedantigen.

In some embodiments, the carbon of —(═E)— is not directly bonded to anaryl group. In some embodiments, the carbon of —C(═E)— is not directlybonded to a phenyl group.

In one embodiment, the disclosure provides antigen-bindingcompound-conjugate represented by formula (I), wherein Z₂ is representedby the following structural formula:

—Z_(2A)-Z_(2B)-Z_(2C)-Z_(2D)—,

wherein: Z_(2A), Z_(2B), Z_(2C) and Z_(2D) are each independentlyabsent, an amino acid, a peptide having 2-20 amino acids, an alkyl, analkynyl, an alkenyl, a cycloalkyl, an aryl, a heteroaryl, aheterocyclyl, —CR₅R₆—, —O—, —C(═O)—, —O—C(═O)—, —C(═O)—O—, —O—C(═O)—O—,—C(═O)—(CH_(x))_(p1)—, —C(═O)—O—(CH_(x))_(p1)—, —(CH_(x))_(p1)—C(═O)—,—(CH_(x))_(p1)—C(═O)—O—, —(O—(CH₂)_(p2)—)_(p3)—, ((CH₂)_(p2)—O—)_(p3)—,—C(═S)—, —C(═S)—S—, —C(═S)—NH—, —S—C(═S)—, —S—C(═S)—S—, —S—, —SO—,—SO₂—, —NR₄—, —N(R₄)—C(═O)—N(R₈)—, —N(R₄)—C(═O)O—, —N(R₄)—C(═O)—,—C(═O)—N(R₄)—, —C(═O)—N(R₄)—C(═O)—, —O—C(═O)—N(R₄), —O—C(═S)—N(R₄)—, or—C(═S)—N(R₄)—, wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl,heteroaryl, and heterocyclyl are optionally substituted and R₄, R₅, R₆and R₈ are each independently H or a substituted or unsubstituted:alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl; and Z_(i) isrepresented by the following structural formula:

—Z_(1A)-Z_(1B)-Z_(1C)-Z_(1D)—,

wherein: Z_(1A), Z_(1B), Z_(1C) and Z_(1D) are each independentlyabsent, an amino acid, a peptide having 2-20 amino acids, an alkyl, analkynyl, an alkenyl, a cycloalkyl, an aryl, a heteroaryl, aheterocyclyl, —CR₅R₆—, —O—, —C(═O)—, —O—C(═O)—, —C(═O)—O—, —O—C(═O)—O—,—C(═O)—(CH_(x))_(p1)—, —C(═O)—O—(CH_(x))_(p1)—, —(CH_(x))_(p1)—C(═O)—,—(CH_(x))_(p1)—C(═O)—O—, —(O—(CH₂)_(p2)—)_(p3)—, —((CH₂)_(p2)—O-)_(p3)—,—C(═S)—, —C(═S)—S—, —C(═S)—NH—, —S—C(═S)—, —S—C(═S)—S—, —S—, —SO—,—SO₂—, NR₄—, —N(R₄)—C(═O)—N(R₈)—, —N(R₄)—C(═O)O—, —N(R₄)—C(═O)—,—C(═O)—N(R₄)—, C(═O)—N(R₄)—C(═O)—, —O—C(═O)—N(R₄), —O—C(═S)—N(R₄)—, or—C(═S)—N(R₄)—, wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl,heteroaryl, and heterocyclyl are optionally substituted and R₄, R₅, R₆and R₈ are each independently H or a substituted or unsubstituted:alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl; p1, p2 andp3 are each independently 0, or an integer from 1 to 100; and x is 0, 1or 2.

In an embodiment, the disclosure provides a compound of formula (I)represented by the following structure (I)(b):

(I)(b).

In an embodiment, the disclosure provides the antigen-bindingcompound-conjugate represented by formula (I)(b), wherein IC1 is a firstheavy chain of an antibody or antigen-binding portion thereof; and IC2is a second heavy chain of the antibody or antigen-binding portionthereof. In a further embodiment, the disclosure provides theantigen-binding compound-conjugate represented by formula (I)(b),wherein the antigen-binding compound-conjugate is substantially stableunder reducing conditions.

In an embodiment, the disclosure provides the antigen-bindingcompound-conjugate represented by formula (I)(b), wherein IC1 is a heavychain of an antibody or antigen-binding portion thereof; and IC2 is alight chain of the antibody or antigen-binding portion thereof. In afurther embodiment, the disclosure provides the antigen-bindingcompound-conjugate represented by formula (I)(b), wherein theantigen-binding compound-conjugate is substantially stable underreducing conditions.

In an aspect, the disclosure also provides a multimeric immunoglobulinconjugate having the structure as set forth in FIG. 1):

wherein one or more M1, M2, M3, and/or M4 are each independently absent(i.e., the adjacent S atoms are directly connected to one another via adisulfide bond), or have the structure represented by formula (II)

wherein LC1 is a first antibody light chain, LC2 is a second antibodylight chain, HC1 is a first antibody heavy chain, and HC2 is a secondantibody heavy chain;

wherein LC1, LC2, HC1 and/or HC2 comprise at least one antigen-bindingdomain; and E is O, S, NR₄, or CR₅R₆; D is a biologically activemolecule; A is absent, a natural or non-natural amino acid, or a peptidecomprising 2-20 amino acids; W is absent, —O—, —S—, —CR₅R₆—, or —NR₄—; Xis absent, aryl, heteroaryl, cycloalkyl, heterocyclyl, wherein aryl,heteroaryl, cycloalkyl, and heterocyclyl are optionally substituted; andY is absent,

wherein A_(l), A₃, R₁ and R₃ are each independently absent, an aminoacid, a peptide having 2-20 amino acids, an alkyl, an alkynyl, analkenyl, a cycloalkyl, an aryl, a heteroaryl, a heterocyclyl, —CR₅R₆—,—O—, —C(═O)—, —O—C(═O)—, —C(═O)—O—, —O—C(═O)—O—, —C(═O)—(CH_(x))_(p1)—,—C(═O)—O—(CH_(x))_(p1)—, —(CH_(x))_(p1)—C(═O)—, —(CH_(x))_(p1)—C(═O)—O—,—O—(CH₂)_(p2)—)_(p3)—, —((CH₂)_(p2)—O—)_(p3), —C(═S)—, —C(═S)—S—,—S—C(═S)—, —C(═S)—NH—, —S—C(═S)—S—, —S—, —SO—, —SO₂, NR₄,—N(R₄)—C(═O)—N(R₈)—, —N(R₄)—C(═O)O—, —N(R₄)—C(═O)—, —C(═O)—N(R₄)—,—C(═O)—N(R₄)—C(═O)—, —O—C(═O)—NR₄—,

wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, andheterocyclyl are optionally substituted; A₄ and A₅ are eachindependently —O—, —S—, —NR₁₈—, or —CR₅R₆—; R₁₇ is O, S, NR₁₈, or CR₅R₆;R₁₈ is H, alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl,heterocyclyl, or acyl, wherein alkyl, alkynyl, alkenyl, cycloalkyl,aryl, heteroaryl, heterocyclyl, and acyl are optionally substituted; R₄,R₅, R₆ and R₈ are each independently H, or a substituted orunsubstituted: alkyl, alkenyl, alkynyl, aryl, heteroaryl, orheterocyclyl; p1, p2 and p3 are each independently 0, or an integer from1 to 100; and x is 0, 1 or 2.

In an embodiment, the disclosure provides a multimeric immunoglobulinconjugate having the structure as set forth in FIG. 1), wherein Z₂ isrepresented by the following structural formula:

—Z_(2A)-Z_(2B)-Z_(2C)-Z_(2D)—,

wherein: Z_(2A), Z_(2B), Z_(2C) and Z_(2D) are each independentlyabsent, an amino acid, a peptide having 2-20 amino acids, an alkyl, analkynyl, an alkenyl, a cycloalkyl, an aryl, a heteroaryl, aheterocyclyl, —CR₅R₆—, —O—, —C(═O)—, —O—C(═O)—, —C(═O)—O—, —O—C(═O)—O—,—C(═O)—(CH_(x))_(p1)—, —C(═O)—O—(CH_(x))_(p1)—, —C(═O)—,—(CH_(x))_(p1)—C(═O)—O—, —(O—(CH₂)_(p2)—)_(p3)—, —((CH₂)_(p2)—O—)_(p3)—,—C(═S)—, —C(═S)—S—, —C(═S)—NH—, —S—C(═S)—, —S—C(═S)—S—, —S—, —SO—, —SO₂,—NR₄—, —N(R₄)—C(═O)—N(R₈)—, —N(R₄)—C(═O)—O—, —N(R₄)—C(═O)—,—C(═O)-N(R₄)—, —C(═O)-N(R₄)-C(═O)—, —O—C(═O)-N(R₄), —O—C(═S)-N(R₄)—, or—C(═S)-N(R₄)—, wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl,heteroaryl, and heterocyclyl are optionally substituted and R₄, R₅, R₆and R₈ are each independently H, or a substituted or unsubstituted:alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl; and Z₁ isrepresented by the following structural formula:

—Z_(1A)-Z_(1B)-Z_(1C)-Z_(1D)—,

wherein: Z_(1A), Z_(1B), Z_(1C) and Z_(1D) are each independentlyabsent, an amino acid, a peptide having 2-20 amino acids, an alkyl, analkynyl, an alkenyl, a cycloalkyl, an aryl, a heteroaryl, aheterocyclyl, —CR₅R₆—, —O—, —C(═O)—, —O—C(═O)—, —C(═O)—O—, —O—C(═O)—O—,—C(═O)—(CH_(x))_(p1)—, —C(═O)—O—(CH_(x))_(p1)—, —(CH_(x))_(p1)-—(═O)—,—(CH_(x))_(p1)—C(═O)—O—, —O—(CH₂)_(p2)—)_(p3)—, ((CH₂)_(p2)—O—)_(p3)—,—C(═S)—, —C(═S)—S—, —C(═S)—NH—, —S—C(═S)—, —S—C(═S) —S—, —S—, —SO—,—SO₂—, —NR₄—, —N(R₄)—C(═O)—N(R₈)—, —N(R₄)—C(═O)O—, —N(R₄)—C(═O)—,—C(═O)—N(R₄)—, C(═O)—N(R₄)—C(═O)—, —O—C(═O)—N(R₄), —O—C(═S)—N(R₄)—, or—C(═S)—N(R₄)—, wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl,heteroaryl, and heterocyclyl are optionally substituted and R₄, R₅, R₆and R₈ are each independently H or a substituted or unsubstituted:alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl; p1, p2 andp3 are each independently 0, or an integer from 1 to 100; and x is 0, 1or 2.

In an embodiment, the disclosure provides a multimeric immunoglobulinconjugate having the structure as set forth in FIG. 1) wherein theconjugate is substantially stable under reducing conditions. In afurther embodiment, the disclosure provides a multimeric immunoglobulinconjugate having the structure as set forth in FIG. 1) wherein theconjugate specifically binds a tumor-associated antigen.

In an embodiment, the disclosure provides a multimeric immunoglobulinconjugate having the structure as set forth in FIG. 1) wherein theconjugate specifically binds a tumor-associated antigen.

In an embodiment, the formula (II) is represented by the followingstructure (II)(b):

Provided herein are compounds of the following formula:

IG(SP^(A)-A-W-X-Y-Z₁-D)_(x)

wherein:

IG is an antigen-binding immunoglobulin;

x is an integer from 1 to 4;

SP^(A) is:

wherein: R^(N) is a hydrogen atom or alkyl; R^(M) is alkyl; the twobonds represented by

are bonds to cysteines of the immunoglobulin and b is an integer from 2to 8; and A, W, X, Y, Z₁, and D are as defined herein.

In some embodiments, the compound has the following structure:

IG(SP^(A)-A-D)_(x)

wherein: IG is an antigen-binding immunoglobulin; x is an integer from 1to 4;

SP^(A) is:

wherein: R^(N) is a hydrogen atom or alkyl; R^(M) is alkyl; the twobonds represented by

are bonds to cysteines of the immunoglobulin; and b is an integer from 2to 8; and A is as defined elsewhere herein.

In some embodiments, A is a dipeptide.

In some embodiments, SP^(A) is:

In some embodiments, SP^(A) is:

In some embodiments, SP^(A) is:

In some embodiments, SP^(A) is:

In some embodiments, SP^(A)-A— is:

wherein:

the two bonds represented by are bonds to cysteines of theimmunoglobulin; and b is an integer from 2 to 8.

In some embodiments, SP^(A)-A— is:

wherein: the two bonds represented by

are bonds to cysteines of the immunoglobulin; and b is an integer from 2to 8.

In some embodiments, SP^(A)-A— is:

wherein:

R^(N) is a hydrogen atom or alkyl;

R^(M) is alkyl;

the two bonds represented by

are bonds to cysteienes of the immunoglobulin; and b is an integer from2 to 8.

In some embodiments, SP^(A)-A— is:

In some embodiments, SP^(A)-A— is:

In some embodiments, SP^(A)-A— is:

In some embodiments, SPA-A— is:

Provided herein are also antibody-drug conjugates comprising anantibody, or antigen binding fragment thereof, wherein the antibody orantigen binding fragment thereof is conjugated to at least one moiety ofFormula (A):

wherein:

Ab-S1 is a bond to a cysteine sulfur atom of the antibody or antigenbinding fragment thereof; Ab-S2 is a bond to a cysteine sulfur atom ofthe antibody or antigen binding fragment thereof; X is —N(R^(A))— or—O—;

wherein R^(A) is a hydrogen atom or alkyl; R^(N) and R^(M) are each,independently, a hydrogen atom or alkyl; A is absent, i.e., a bond, or aspacer comprising a peptide,

wherein the peptide comprises 2-20 amino acids; D is a biologicallyactive molecule; and b is an integer from 2 to 8.

In some embodiments, the antibody or antigen binding fragment thereofcomprises at least one moiety of Formula (A) wherein Ab-S1 is a bond toa cysteine sulfur atom of a first heavy chain of the antibody or antigenbinding fragment thereof and AB-S2 is a bond to a cysteine sulfur atomof a second heavy chain of the antibody or antigen binding fragmentthereof.

In some embodiments, the antibody or antigen binding fragment thereofcomprises at least one moiety of Formula (A) wherein Ab-S1 is a bond toa cysteine sulfur atom of a light chain of the antibody or antigenbinding fragment thereof and AB-S2 is a bond to a cysteine sulfur atomof a heavy chain of the antibody of antigen binding fragment thereof.

In some embodiments, the antibody or antigen binding fragment thereofcomprises:

(i) at least one moiety of Formula (A) wherein Ab-S1 is a bond to acysteine sulfur atom of a first heavy chain of the antibody or antigenbinding fragment thereof and AB-S2 is a bond to a cysteine sulfur atomof a second heavy chain of the antibody or antigen binding fragmentthereof; and

i) (ii) at least one moiety of Formula (A) wherein Ab-S1 is a bond to acysteine sulfur atom of a light chain of the antibody or antigen bindingfragment thereof and AB-S2 is a bond to a cysteine sulfur atom of aheavy chain of the antibody of antigen binding fragment thereof.

In some embodiments, the antibody or antigen binding fragment thereofcomprises two moieties of Formula (A) wherein Ab-S1 is a bond to acysteine sulfur atom of a first heavy chain of the antibody or antigenbinding fragment thereof and AB-S2 is a bond to a cysteine sulfur atomof a second heavy chain of the antibody or antigen binding fragmentthereof.

In some embodiments, the antibody or antigen binding fragment thereofcomprises:

(i) two moieties of Formula (A) wherein Ab-S1 is a bond to a cysteinesulfur atom of a first heavy chain of the antibody or antigen bindingfragment thereof and AB-S2 is a bond to a cysteine sulfur atom of asecond heavy chain of the antibody or antigen binding fragment thereof;and

(ii) two moieties of Formula (A) wherein Ab-S1 is a bond to a cysteinesulfur atom of a light chain of the antibody or antigen binding fragmentthereof and AB-S2 is a bond to a cysteine sulfur atom of a heavy chainof the antibody of antigen binding fragment thereof.

In some embodiments, the antibody drug conjugate comprises an antibody.

In some embodiments, the antibody is an anti-PRLR antibody.

In some embodiments, X is —N(R^(A))—. In certain embodiments, R^(A) is ahydrogen atom.

In some embodiments, X is —O—.

In some embodiments, R^(N) and R^(M) are both hydrogen atoms. In someembodiments, R^(N) is a hydrogen atom and R^(M) is alkyl. In someembodiments, R^(N) and R^(M) are both alkyl.

In some embodiments, X is —O— and R^(N) and R^(M) are both hydrogenatoms. In some embodiments, X is —O—, R^(N) is a hydrogen atom, andR^(M) is alkyl. In some embodiments, X is —O— and R^(N) and R^(M) areboth alkyl. In some embodiments, X is —O—, R^(N) is a hydrogen atom, andR^(M) is methyl. In some embodiments, X is —O— and R^(N) and R^(M) areboth methyl.

In some embodiments, b is an integer from 3-6. In some embodiments, b is4. In some embodiments, R^(N) and R^(M) are both hydrogen atoms and b is4.

In some embodiments, X is —O—, R^(N) and R^(M) are both hydrogen atoms,and b is 4. In some embodiments, X is —O—, R^(N) and R^(M) are bothalkyl, and b is 4. In some embodiments, X is —O—, R^(N) and R^(M) areboth methyl, and b is 4. In some embodiments, X is —O—, R^(N) is alkyl,R^(M) is a hydrogen atom, and b is 4.

In some embodiments, X is —N(H)—, R^(N) and R^(M) are both hydrogenatoms, and b is 4.

In some embodiments, R^(A), R^(N) and R^(M) are each, independently is ahydrogen atom or C₁₋₆ alkyl.

In some embodiments, A is a spacer comprising a dipeptide. In someembodiments, the dipeptide is valine-citrulline. In some embodiments, Ais:

wherein

is the bond to D, and AA₁ and AA₂ are each, independently, an aminoacid.

In some embodiments, AA¹-AA² is: valine-citrulline, citrulline-valine,lysine-phenylalanine, phenylalanine-lysine, valine-asparagine,asparagine-valine, threonine-asparagine, serine-asparagine,asparagine-serine, phenylalanine-asparagine, asparagine-phenylalanine,leucine-asparagine, asparagine-leucine, isoleucine-asparagine,asparagine-isoleucine, glycine-asparagine, asparagine-glycine, glutamicacid- asparagine, asparagine-glutamic acid, citrulline-asparagine,asparagine-citrulline, alanine-asparagine, or asparagine-alanine.

In some embodiments, A is:

wherein

is the bond to D and R^(AA1) and R^(AA2) are each, independently, anamino acid side chain. As used herein, “amino acid side chain” refersthe monovalent non-hydrogen substituent bonded to the α-carbon of anα-amino acid, including natural and non-natural amino acids. Exemplaryamino acid side chains include, but are not limited to, the α-carbonsubstituent of alanine, valine, leucine, isoleucine, methionine,tryptophan, phenylalanine, proline, serine, threonine, cysteine,tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine,arginine, histidine, and citrulline.

In some embodiments, A is:

wherein

is the bond to D.

In some embodiments, A is absent.

In some embodiments, D is an auristatin or maytansinoid.

In some embodiments, D is an auristatin, wherein the auristatin is MMAE,MMAD, or MMAF.

In some embodiments, D is MMAF.

In some embodiments, D is a maytansinoid

In some embodiments, A is a spacer comprising a peptide comprising 2-20amino acids and D is a maytansinoid.

In some embodiments, A is absent and D is an auristatin.

In some embodiments, A is represented by formula (I)(a) as biologicallyactive macrolide:

wherein A₆, A₇, A₈, A₉ are each independently absent, an amino acid,N-alkyl amino acid, a peptide having 2-20 amino acids, an alkyl, analkenyl, an alkynyl, a cycloalkyl, an aryl, a heteroaryl, aheterocyclyl, —CR₅R₆—, —O—, —C(═O)—, —O—C(═O)—, —C(═O)—O—, —O—C(═O)—O—,—C(═O)—(CH_(x))_(p1)—, —C(═O)—O—(CH_(x))_(p1), —(CH_(x))_(p1)—C(═O)—,—(CH_(x))_(p1)—C(═O)—O—, —(O—(CH₂)_(p2))_(p2)—)_(p3)—,—((CH₂)_(p2)—O—)_(p3), —C(═S)—, —C(═S)—NH—, —C(═S)—S—, —S—C(═S)—,—S—C(═S) —S—, —S—, —SO—, —SO₂—, —NR₄—, —N(R₄)—C(═O)—N(R₈)—,—N(R₄)—C(═O)—, —N(R₄)—C(═O)—, —C(═O)—N(R₄)—, —C(═O)—N(R₄)—C(═O)—,—O—C(═O)—NR₄, further wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl,heteroaryl, and heterocyclyl are optionally substituted; and R₄, R₅, R₆and R₈ are each independently H, or a substituted or unsubstituted:alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl; p1, p2 andp3 are each independently 0, or an integer from 1 to 100; and x is 0, 1or 2.

In some embodiments, D is DM1 or DM4.

In some embodiments, D is:

In some embodiments, D is:

In some embodiments, the moiety of Formula (A) is:

In some embodiments, the moiety of Formula (A) is:

In some embodiments, the moiety of Formula (A) is:

wherein R^(N) and R^(M) are, independently, a hydrogen atom or C₁₋₆alkyl.

In some embodiments, the moiety of Formula (A) is:

In some embodiments, the moiety of Formula (A) is:

In some embodiments, the moiety of Formula (A) is:

wherein D is a maytansinoid.

In some embodiments, the moiety of Formula (A) is:

wherein D is a maytansinoid.

In some embodiments, the moiety of Formula (A) is:

In some embodiments, the moiety of Formula (A) is:

In some embodiments, the moiety of Formula (A) is:

wherein D is an auristatin.

In some embodiments, the moiety of Formula (A) is:

wherein D is an auristatin.

In some embodiments, the antibody or antigen binding fragment thereoffurther comprises a moiety of Formula (B1) or (B2):

In some embodiments, moiety of Formula (A) is:

III. METHODS OF SYNTHESIZING CONJUGATES

A. Conjugation Methods

The disclosure provides a method for preparing a multimericimmunoglobulin conjugate having the structure as set forth in FIG. 1):

wherein one or more M₁, M₂, M₃, and/or M₄ are each independently absent(i.e., the adjacent S atoms are directly connected to one another via adisulfide bond), or have the structure represented by formula (II)

wherein LC1 is a first antibody light chain, LC2 is a second antibodylight chain, HC1 is a first antibody heavy chain, and HC2 is a secondantibody heavy chain;

wherein LC1, LC2, HC1 and/or HC2 comprise at least one antigen-bindingdomain; E is O, S, NR₄, or CR₅R₆; D is a biologically active molecule;Z₁ and Z₂ are each independently absent or a spacer; A is absent, anatural or non-natural amino acid, or a peptide comprising 2-20 aminoacids; W is absent, —O—, —S—, —CR₅R₆—, or —NR₄—; X is absent, aryl,heteroaryl, cycloalkyl, heterocyclyl,

wherein aryl, heteroaryl, cycloalkyl, and heterocyclyl are optionallysubstituted; Y is absent,

wherein A₁, A₃, R₁ and R₃ are each independently absent, an amino acid,a peptide having 2-20 amino acids, an alkyl, an alkynyl, an alkenyl, acycloalkyl, an aryl, a heteroaryl, a heterocyclyl, —CR₅R₆—, —O—,—C(═O)—, —O—C(═O)—, —C(═O)—O—, —O—C(═O)—O—, —C(═O)—(CH_(x))_(p1)—,—C(═O)—O—(CH_(x))_(p1)—, —(CH_(x))_(p1)—C(═O)—, —(CH_(x))_(p1)—C(═O)—O—,—O—(CH₂)_(p2)—)_(p3)—, —((CH₂)_(p2)—O—)_(p3)—, —C(═S)—, —C(═S)—S—,—S—C(═S)—, —C(═S)—NH—, —S—C(═S)—S—, —S—, —SO—, —SO₂—, —NR₄—,—N(R₄)—C(═O)—N(R₈)—, —N(R₄)—C(═O)O—, —N(R₄)—C(═O)—, —C(═O)—N(R₄)—,—C(═O)—N(R₄)—C(═O)—, or —O—C(═O)—NR₄—,

wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, andheterocyclyl are optionally substituted; A₄ and A₅ are eachindependently —O—, —S—, —NR₁₈—, or —CR₅R₆—; R₁₇ is O, S, NR₁₈, or CR₅R₆;R₁₈ is H, alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl,heterocyclyl, or acyl, wherein alkyl, alkynyl, alkenyl, cycloalkyl,aryl, heteroaryl, heterocyclyl, and acyl are optionally substituted; R₄,R₅, R₆ and R₈ are each independently H or a substituted orunsubstituted: alkyl, alkenyl, alkynyl, aryl, heteroaryl, orheterocyclyl; p1, p2 and p3 are each independently 0, or an integer from1 to 100; x is 0, 1 or 2; the method comprising the steps of (a)reducing one or more disulfide bond between cysteine residues present ina multimeric immunoglobulin forming two sulfhydryl groups; and (b)conjugating a reagent that forms a covalent bond with the sulfhydrylgroups derived from step (a) wherein the reagent is represented byformula (III):

further wherein LG is a leaving group; E is O, S, NR₄, or CR₅R₆; Z₁ andZ₂ are each independently absent or a spacer; D is absent or abiologically active molecule; A is absent, a natural or non-naturalamino acid, or a peptide comprising 2-20 amino acids; W is absent, —O—,—S—, —CR₅R₆—, or —NR₄—; X is absent, aryl, heteroaryl, cycloalkyl,heterocyclyl, wherein aryl, heteroaryl, cycloalkyl, and heterocyclyl areoptionally substituted; and Y is absent,

wherein A₁, A₃, R₁ and R₃ are each independently absent, an amino acid,a peptide having 2-20 amino acids, an alkyl, an alkynyl, an alkenyl, acycloalkyl, an aryl, a heteroaryl, a heterocyclyl, —CR₅R₆—, —O—,—C(═O)—, —O—C(═O)—, —C(═O)—O—, —O—C(═O)—O—, —C(═O)—(CH_(x))_(p1)—,—C(═O)—O—(CH_(x))_(p1)—, —(CH_(x))_(p1)—C(═O)—, —(CH_(x))_(p1)—C(═O)—O—,—(O—(CH₂)_(p2)—)_(p3)—, ((CH₂)_(p2)—O—)_(p3)—, —C(═S)—, —C(═S)—S—,—S—C(═S)—, —C(═S)—NH—, —S—C(═S)—S—, —S—, —SO—, —SO₂—, —NR₄—,—N(R₄)C(═O)—N(R₈)—, —N(R₄)—C(═O)O—, —N(R₄)—C(═O)—, —C(═O)—N(R₄)—,—C(═O)—N(R₄)—C(═O)—, or —OC(═O)—NR₄—, wherein alkyl, alkynyl, alkenyl,cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionallysubstituted; A₄ and A₅ are each independently —O—, —S—, —NR₁₈—, or—CR₅R₆—; R₁₇ is O, S, NR₁₈, or CR₅R₆; R₁₈ is H, alkyl, alkynyl, alkenyl,cycloalkyl, aryl, heteroaryl, heterocyclyl, or acyl, wherein alkyl,alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, and acylare optionally substituted; R₄, R₅, R₆ and R₈ are each independently H,or a substituted or unsubstituted: alkyl, alkenyl, alkynyl, aryl,heteroaryl, or heterocyclyl; p1, p2 and p3 are each independently 0, oran integer from 1 to 100; and x is 0, 1 or 2.

In an embodiment, the method for preparing a multimeric immunoglobulinconjugate further comprises purifying the product obtained in step (b)by chromatography, dialysis, ultra filtration, and/or tangential flowfiltration.

In an embodiment, the disclosure provides a method for preparing amultimeric immunoglobulin conjugate, wherein Z₂ is represented by thefollowing structural formula:

—Z_(2A)-Z_(2B)-Z_(2C)-Z_(2D)—,

wherein: Z_(2A), Z_(2B), Z_(2C) and Z_(2D) are each independentlyabsent, an amino acid, a peptide having 2-20 amino acids, an alkyl, analkynyl, an alkenyl, a cycloalkyl, an aryl, a heteroaryl, aheterocyclyl, —CR₅R₆—, —O—, —C(═O)—, —O—C(═O)—, —C(═O)—O—, —O—C(═O—)—O—,—C(═O)—(CH_(x))_(p1)—, —C(═O)—O—(CH_(x))_(p1), —(CH_(x))_(p1)—C(═O)—,—(CH_(x))_(p1)—C(═O)—O—, —(O—(CH₂)_(p2)—)_(p3)—,((CH₂)_(p2)—O—)_(p3)—,—C(═S)—, —C(═S)—S—, —C(═S)—NH—, —S—C(═S)—, —S—C(═S) —S—, —S—, —SO—,—SO₂—, —NR₄—, —N(R₄)—C(═O)—N(R₈)—, —N(R₄)—C(═O)O—, N(R₄)—C(═O)—,—C(═O)—N(R₄)—, —C(═O)—N(R₄)—C(═O)—, —O—C(═O)—N(R₄), —O—C(═S)—N(R₄)—, or—C(═S)—N(R₄)—, wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl,heteroaryl, and heterocyclyl are optionally substituted and R₄, R₅, R₆and R₈ are each independently H, or a substituted or unsubstituted:alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl; Z₁ isrepresented by the following structural formula:

—Z_(1A)-Z_(1B)-Z_(1C)-Z_(1D)—,

wherein: Z_(1A), Z_(1B), Z_(1C) and Z_(1D) are each independentlyabsent, an amino acid, a peptide having 2-20 amino acids, an alkyl, analkynyl, an alkenyl, a cycloalkyl, an aryl, a heteroaryl, aheterocyclyl, —CR₅R₆—, —O—, —C(═O)—, —O—C(═O)—, —C(═O)—O—, —O—C(═O)—O—,—C(═O)—(CH_(x))_(p1)—, —C(═O)—O—(CH_(x))_(p1)—, —(CH_(x))_(p1)—C(═O)—O—,—O—(CH₂)_(p2)—)_(p3)—, —((CH₂)_(p2)—O—)_(p3)—, —((CH₂)_(p2)—O—)_(p3)—,—C(═S)—, —C(═S)—S—, —C(═S)—NH—, —S—C(═S)—, —S—C(═S)—S—, —S—, —SO—,—SO₂—, —NR₄—, —N(R₄)—C(═O)—N(R₈)—, —N(R₄)—C(═O)O—, —N(R₄)—C(═O)—,—C(═O)—N(R₄)—, C(═O)—N(R₄)—C(═O)—, —O—C(═O)—N(R₄), —O—C(═S)—N(R₄)—, or—C(═S)—N(R₄)—, wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl,heteroaryl, and heterocyclyl are optionally substituted and R₄, R₅, R₆and R₈ are each independently H or a substituted or unsubstituted:alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl; p1, p2 andp3 are each independently 0, or an integer from 1 to 100; and x is 0, 1or 2.

In a further embodiment, the disclosure provides the method forpreparing a multimeric immunoglobulin conjugate wherein the leavinggroup in the reagent represented by structural formula (III) is ahalogen, tosyl, mesyl, OAc, OMe, triflate, nitrate, thiolate, phosphate,carboxylate, or phenoxide. In a further embodiment, the leaving group isbromo.

In an embodiment, the disclosure provides the method for preparing amultimeric immunoglobulin conjugate wherein formula (II) is representedby the following structure (II)(b):

In a further embodiment, the disclosure provides the method forpreparing a multimeric immunoglobulin conjugate wherein formula (III) isrepresented by the following structure (III)(b):

B. Linker-Payload Reagents

The disclosure also relates to a reagent represented by the followingstructural formula (III):

wherein: LG is a leaving group; E is O, S, NR₄, or CR₅R₆; Z₁ and Z₂ areeach independently absent or a spacer; D is absent or a biologicallyactive molecule; A is absent, a natural or non-natural amino acid, or apeptide comprising 2-20 amino acids; W is absent, —O—, —S—, —CR₅R₆—, or—NR₄—; X is absent, aryl, heteroaryl, cycloalkyl, or heterocyclyl,wherein aryl, heteroaryl, cycloalkyl, and heterocyclyl are optionallysubstituted; and Y is absent,

wherein A₁, A₃, R₁ and R₃ are each independently absent, an amino acid,a peptide having 2-20 amino acids, an alkyl, an alkynyl, an alkenyl, acycloalkyl, an aryl, a heteroaryl, a heterocyclyl, —CR₅R₆—, —O—,—C(═O)—, —O—C(═O)—, —C(═O)—O—, —O—C(═O)—O—, —C(═O)—(CH_(x))_(p1)—,—C(═O)—O—(CH_(x))_(p1)—, —(CH_(x))_(p1)—C(═O)—, —(CH_(x))_(p1)—C(═O)—O—,—(O—CH₂)_(p2)—)_(p3)—, —((CH₂)_(p2)—O—)_(p3)—, —C(═S)—, —C(═S)—S—,—S—C(═S)—, —C(═S)—NH—, —S—C(═S)—S—, —S—, —SO—, —SO₂—, —NR₄—,—N(R₄)—C(═O)—N(R₈)—, —N(R₄)—C(═O)O—, —N(R₄)—C(═O)—, —C(═O)—N(R₄)—,—C(═O)—N(R₄)—C(═O)—, or —O—C(═O)—NR₄—, wherein alkyl, alkynyl, alkenyl,cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionallysubstituted; A₄ and A₅ are each independently —O—, —S—, —NR₁₈—, or—CR₅R₆—; R₁₇ is O, S, NR₁₈, or CR₅R₆; R₁₈ is H, alkyl, alkynyl, alkenyl,cycloalkyl, aryl, heteroaryl, heterocyclyl, or acyl, wherein alkyl,alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, and acylare optionally substituted; R₄, R₅, R₆ and R₈ are each independently H,or a substituted or unsubstituted: alkyl, alkenyl, alkynyl, aryl,heteroaryl, or heterocyclyl; p1, p2 and p3 are each independently 0, oran integer from 1 to 100; and x is 0, 1 or 2.

In an embodiment, the disclosure provides a reagent represented by thestructural formula (III), wherein Z₂ is represented by the followingstructural formula:

—Z_(2A)-Z_(2B)-Z_(2C)-Z_(2D)—,

wherein: Z_(2A), Z_(2B), Z_(2C) and Z_(2D) are each independentlyabsent, an amino acid, a peptide having 2-20 amino acids, an alkyl, analkynyl, an alkenyl, a cycloalkyl, an aryl, a heteroaryl, aheterocyclyl, —CR₅R₆—, —O—, —C(═O)—, —O—C(═O)—, —C(═O)—O—, —O—C(═O))—O—,—C(═O)—(CH_(x))_(p1)—, —C(═O)—O—(CH_(x))_(p1)—, —(CH_(x))_(p1)—C(═O)—,—(CH_(x))_(p1)—C(═O)—O—, —(O—(CH₂)_(p2)—)_(p2)—,—((CH₂)_(p2))_(p2)—O—)_(p3)—, —C(═S)—, —C(═S)—S—, —C(═S)—NH—, —S—C(═S)—,—S—C(═S)—S—, —S—, —SO—, —SO₂—, —NR₄—, —N(R₄)—C(═O)—N(R₈)—,—N(R₄)—C(═O)—, —(C═O)—N(R₄)—, —C(═O)—N(R₄)—, —C(═O)—, —O—C(═O)—N(R₄),—O—C(═S)—N(R₄)—, or —C(═S)—N(R₄)—, wherein alkyl, alkynyl, alkenyl,cycloalkyl, aryl, heteroaryl, and heterocyclyl are optionallysubstituted and R₄, R₅, R₆ and R₈ are each independently H, or asubstituted or unsubstituted: alkyl, alkenyl, alkynyl, aryl, heteroaryl,or heterocyclyl; and Z₁is represented by the following structuralformula:

—Z_(1A)-Z_(1B)-Z_(1C)-Z_(1D)—,

wherein: Z_(1A), Z_(1B), Z_(1C) and Z_(1D) are each independentlyabsent, an amino acid, a peptide having 2-20 amino acids, an alkyl, analkynyl, an alkenyl, a cycloalkyl, an aryl, a heteroaryl, aheterocyclyl, —CR₅R₆—, —O—, —C(═O)—, —O—C(═O)—, —C(═O)—O—, —O—C(═O)—O—,—C(═O)—(CH_(x))_(p1)—, —C(═O)—O—(CH_(x))_(p1)—, —(CH_(x))_(p1)—C(═O)—,—(CH_(x))_(p1)—C(═O)—O—, —(O—(CH₂)_(p2)—)_(p3), —((CH₂)_(p2)—O—)_(p3)—,—C(═S)—, —C(═S)—S—, —C(═S)—NH—, —S—C(═S)—, —S—C(═S)—S—, —S—, —SO—,—SO₂—, —NR₄—, —N(R₄)—C(═O)—N(R₈)—, —N(R₄)—C(═O)O—, —N(R₄)—C(═O)—,—C(═O)—N(R₄)—, C(═O)—N(R₄)—C(═O)——O—C(═O)—N(R₄), —O—C(═S)—N(R₄)—, or—C(═S)—N(R₄)—, wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl,heteroaryl, and heterocyclyl are optionally substituted and R₄, R₅, R₆and R₈ are each independently H, or a substituted or unsubstituted:alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclyl; p1, p2 andp3 are each independently 0, or an integer from 1 to 100; and x is 0, 1or 2.

In a further embodiment, the disclosure provides a reagent representedby structural formula (III), wherein the leaving group is a halogen,tosyl, mesyl, OAc, OMe, triflate, nitrate, thiolate, phosphate,carboxylate, phenoxide. In a further embodiment, the disclosure providesa reagent represented by structural formula (III), wherein the leavinggroup is bromo.

In an embodiment, the disclosure provides a reagent of formula (III)represented by the following structure (III)(b):

In another embodiment, the compound has the formula (aa) or (bb):

M-Z₂-A-W-X-Y-Z₁-D  (aa)

M-A-D  (bb)

wherein M is:

wherein X is —O— or —NH— and LG is a leaving group, e.g., Br; and Z₂, A,W, X, Y, Z₁, and D are as defined herein.

In some embodiments, the (i) M-Z2-A- of formula (aa) or (ii) M-A- offormula (bb) is:

wherein b is an integer from 2 to 8, R^(N) is a hydrogen atom or alkyl,and R^(M) is alkyl.

Provided herein are also linker-payload compounds of Formula (L1):

wherein:

R^(U is)

wherein LG¹ and LG², independently at each occurrence, is a leavinggroup; X is —N(R^(A))— or —O—;

wherein R^(A) is a hydrogen atom or alkyl; R^(N) and R^(M) are each,independently, a hydrogen atom or alkyl; A is absent, i.e., a bond, or aspacer comprising a peptide, wherein the peptide comprises 2-20 aminoacids; D is a biologically active molecule; and b is an integer from 2to 8.

In some embodiments, the leaving group is a halogen, tosyl, mesyl, OAc,OMe, triflate, nitrate, thiolate, phosphate, carboxylate, or phenoxide.

In some embodiments, the leaving group is —Br.

In some embodiments, X is —N(R^(A))—. In certain embodiments, R^(A) is ahydrogen atom.

In some embodiments, X is —O—.

In some embodiments, R^(N) and R^(M) are both hydrogen atoms. In someembodiments, R^(N) is a hydrogen atom and R^(M) is alkyl. In someembodiments, R^(N) and R^(M) are both alkyl.

In some embodiments, X is —O— and R^(N) and R^(M) are both hydrogenatoms. In some embodiments, X is —O—, R^(N) is a hydrogen atom, andR^(M) is alkyl. In some embodiments, X is —O—and R^(N) and R^(M) areboth alkyl. In some embodiments, X is —O—, R^(N) is a hydrogen atom, andR^(M) is methyl. In some embodiments, X is —O— and R^(N) and R^(M) areboth methyl.

In some embodiments, b is an integer from 3-6. In some embodiments, b is4. In some embodiments, R^(N) and R^(M) are both hydrogen atoms and b is4.

In some embodiments, X is —O—, R^(N) and R^(M) are both hydrogen atoms,and b is 4. In some embodiments, X is —O—, R^(N) and R^(M) are bothalkyl, and b is 4. In some embodiments, X is —O—, R^(N) and R^(M) areboth methyl, and b is 4. In some embodiments, X is —O—, R^(N) is alkyl,R^(M) is a hydrogen atom, and b is 4.

In some embodiments, X is —N(H)—, R^(N) and R^(M) are both hydrogenatoms, and b is 4.

In some embodiments, A is a spacer comprising a dipeptide. In someembodiments, the dipeptide is valine-citrulline. In some embodiments, Ais:

wherein

is the bond to D.

In some embodiments, A is:

wherein

is the bond to D.

In some embodiments, A is absent.

In some embodiments, D is an auristatin or maytansinoid.

In some embodiments, D is an auristatin, wherein the auristatin is MMAE,MMAD, or MMAF.

In some embodiments, D is MMAF.

In some embodiments, D is a maytansinoid

In some embodiments, A is spacer comprising a peptide comprising 2-20amino acids and D is a maytansinoid.

In some embodiments, A is absent and D is an auristatin.

In some embodiments, A is represented by formula (I)(a) as biologicallyactive macrolide:

wherein A₆, A₇, A₈, A₉ are each independently absent, an amino acid,N-alkyl amino acid, a peptide having 2-20 amino acids, an alkyl, analkenyl, an alkynyl, a cycloalkyl, an aryl, a heteroaryl, aheterocyclyl, —CR₅R₆—, —O—, —C(═O)—, —O—C(═O)—, —C(═O)—O—, —O—C(═O)—O—,—C(═O)—(CH_(x))_(p1)—, —C(═O)—O—(CH_(x))_(p1)—C(═O)—,—(CH_(x))_(p1)—C(═O)—O—, —(O—(CH₂)_(p2)—)_(p3)—, —((CH₂)_(p2)—O—)_(p3)—,—C(═S)—, —C(═S)—NH—, —C(═S)—S—, —S—C(═S)—, —S—C(═S)—S—, —S—, —SO—, —SO₂,—NR₄—, —N(R₄)—C(═O)—N(R₈)—, —N(R₄)—C(═O)O—, —N(R₄)—C(═O)—,—C(═O)—N(R₄)—, —C(═O)—N(R₄)—C(═O)—, —O—C(═O)—NR₄, further wherein alkyl,alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl areoptionally substituted; and R₄, R₅, R₆ and R₈ are each independently H,or a substituted or unsubstituted: alkyl, alkenyl, alkynyl, aryl,heteroaryl, or heterocyclyl; p1, p2 and p3 are each independently 0, oran integer from 1 to 100; and x is 0, 1 or 2.

In some embodiments, D is DM1 or DM4.

In some embodiments, D is:

In some embodiments, D is:

In some embodiments, the compound of Formula (L1) is:

In some embodiments, the compound of Formula (L1) is:

In some embodiments, the compound of Formula (L1) is:

wherein R^(N) and R^(M) are, independently, a hydrogen atom or C₁₋₆alkyl.

In some embodiments, compound of Formula (L1) is:

In some embodiments, the compound of Formula (L1) is:

In some embodiments, the compound of Formula (L1) is:

wherein D is a maytansinoid.

In some embodiments, the compound of Formula (L1) is:

wherein D is a maytansinoid.

In some embodiments, the compound of Formula (L1) is:

In some embodiments, the compound of Formula (L1) is:

In some embodiments, the compound of Formula (L1) is:

wherein D is an auristatin.

In some embodiments, the compound of Formula (L1) is:

wherein D is an auristatin.

In some embodiments, compound of Formula (L1) is:

Linker payloads can be synthesized via carboxylic acid couplingconditions, e.g., as depicted below:

The carboxylic acid can be synthesized from acid halides, e.g., asdepicted below:

IV. PHARMACEUTICAL COMPOSITIONS AND METHODS OF USE

Embodiments here include compositions comprising the conjugatesdescribed herein, e.g., compounds of formula (I), FIG. 1), or formula(III) as well as mixtures thereof. In some aspects the compound isfurther represented by a compound of formula (I)(b), formula (II)(b), orformula (III)(b).

Compositions may be pharmaceutical compositions that further include oneor more pharmaceutically acceptable carriers, diluents, and/orexcipients. In some aspects the pharmaceutical composition is thepharmaceutically acceptable salt of the conjugates described herein,e.g., the compounds of formula (I), FIG. 1), and/or formula (III) ormixtures thereof.

Suitable pharmaceutical acceptable carriers, diluents and excipients arewell known in the art and can be determined by one of ordinary skill inthe art as the clinical situation warrants. Examples of suitablecarriers, diluents and excipients include: buffers for maintenance ofproper composition pH (e.g., citrate buffers, succinate buffers, acetatebuffers, phosphate buffers, lactate buffers, oxalate buffers and thelike), carrier proteins (e.g., human serum albumin), saline, polyols(e.g., trehalose, sucrose, xylitol, sorbitol, and the like), surfactants(e.g., polysorbate 20, polysorbate 80, polyoxolate, and the like),antimicrobials, and antioxidants.

If so desired, the pharmaceutical compositions herein may include asecond or more therapeutic agent (e.g., an adjuvant to the formula (I),FIG. 1), and/or formula (III)). The second therapeutic agent can beincluded in the same composition as the compounds of, e.g., formula (I),FIG. 1), and/or formula (III), or can be administered separately fromthe compounds of, e.g., formula (I), FIG. 1), and/or formula (III) (bytime, or type and location of administration).

One of skill in the art of biologically active molecules will understandthat each of the compounds of, e.g., formula (I), FIG. 1), and/orformula (III) can be modified in such a manner that the resultingcompound still retains specificity and/or activity similar to thestarting compound. In this light, the biologically active molecule (D)of compounds of, e.g., formula (I), FIG. 1), and/or formula (III) caninclude any and all of the biologically active molecules' analogues andderivatives.

In one aspect, the disclosure provides the pharmaceutical compositioncomprising a therapeutically effective amount of a compound of, e.g.,formula (I), FIG. 1), and/or formula (III), or a pharmaceuticallyacceptable salt thereof and one or more pharmaceutically acceptablecarriers, diluents, or excipients.

In another aspect, the disclosure provides pharmaceutical compositioncomprising a therapeutically effective amount of a compound of, e.g.,formula (I)(b), formula (II)(b), and/or formula (III)(b), or apharmaceutically acceptable salt thereof and one or morepharmaceutically acceptable carriers, diluents, or excipients.

As described above, conjugate compounds of, e.g., formula (I), and/orFIG. 1), can be produced with various functional groups such thatattachment of the multimeric immunoglobulin or the multimericantigen-binding compound to the linker and thereby a biologically activemolecule form a covalent conjugate. The multimeric immunoglobulin or themultimeric anti-binding compound specially targets the conjugatecompound to the multimeric immunoglobulin or the multimeric anti-bindingcompound binding partner, typically a polypeptide or other like antigen.In an embodiment, the conjugate is designed to include a multimericimmunoglobulin or the multimeric anti-binding compound having a bindingpartner found on cells undergoing abnormal cell growth or cells involvedin a proliferative disorder. In an embodiment, conjugate compounds of,e.g., formula (I), and/or FIG. 1) have been designed such that eachcompound's linker is catabolized inside the cell bound by the conjugate.As such, delivery of a biologically active molecule through theconjugate embodiments herein allows for delivery of biologically activemolecules that would normally be too toxic to administer conventionally.The embodiments herein allow for highly selective and specific deliveryof these conjugates to cells undergoing abnormal cell growth or cellsinvolved in proliferative disorders (as compared to catabolism outsidethe cell, thereby releasing the biologically active compound into theblood or lymphatic system, for example).

As can be envisioned by one of skill in the art, the covalent conjugatecompounds described herein can also be used to deliver any type ofuseful biologically active molecule and can be selectively targeted toany type of cell population, for example, the conjugate may be used todeliver anti-proliferative drugs to cells undergoing abnormal growth oranti-viral drugs to cells infected with a virus, as long as the selectedmultimeric immunoglobulin or the multimeric anti-binding compoundrecognizes a proper cell binding partner.

In this light, methods of use are provided for the conjugate compoundembodiments described herein.

The pharmaceutical compositions described herein are useful ininhibiting, retarding and/or preventing abnormal cell growth or in thetreatment of various proliferative disorders or disease states inmammals. In typical embodiments the mammal is a human (embodimentsherein will be described in relation to humans). Other mammals includeany mammal that can suffer from a detectable proliferative disorder,including primates, dogs, cats, horses, goats, sheep, cattle, camels,and the like. In addition, it is understood that the conjugate compoundsof the pharmaceutical compositions are designed for selective targetingto the cells undergoing abnormal cell growth or for the treatment of thevarious proliferative disorders or disease states described herein.

As such, embodiments herein include methods of inhibiting abnormal cellgrowth or treatment of a proliferative disorder in a human comprisingadministering to the human a therapeutically effective amount of apharmaceutical composition described herein.

Administration of a therapeutically effective amount of a pharmaceuticalcomposition described herein may be effected in different ways, e.g., byintravenous, intraperitoneal, subcutaneous, intramuscular, topical,intradermal, intranasal, or intrabronchial administration. Thepharmaceutical compositions herein may also be administered directly toan abnormal cell growth site (directly or indirectly contacting theabnormal cell growth) by, for example, biolistic delivery (biolisticdelivery of the pharmaceutical compositions herein to a lung or braintumor, for example). Dosage regiments for administration of thepharmaceutical compositions herein will be determined by the attendinghealth care professional or other person of skill in the art as well asbased on the particular clinical situation. As is well known in thepharmaceutical arts, dosages for any one human, i.e., patient, dependson a number of factors, including patient size, patient's body surfacearea, patient's age and general health, patient's sex, the time androute of administration, and presence of a second therapeutic agent. Insome instances the conjugates described herein, e.g., compounds offormula (I), FIG. 1), and/or formula (III) may be present in amountsbetween 1 μg and 100 mg/kg body weight per dose (note that wherecontinuous infusion is considered as an administration route, as littleas 1 pg/kg body weight per minute may be considered). Pharmaceuticalcompositions can be administered one or more times a day and over aperiod of days, weeks, months, or years.

Treatment of proliferative disorder or disease, for example a tumor,includes methods of reducing a tumor size, causing necrosis or apoptosisin a tumor, killing a tumor, stopping a tumor from increasing in sizeand/or preventing invasiveness or metastasis of a tumor.

Examples of medical conditions that can be treated according to methodsof inhibiting abnormal cell growth, or treating proliferative disordersinclude: malignancy of any type, e.g., cancer of the lung, colon,prostate, kidney, pancreas, liver, ovary, skin, lymphoma, leukemia andthe like; autoimmune diseases, e.g., systemic lupus, rheumatoidarthritis, multiple sclerosis; viral infections, e.g., CMV infection,HIV infection, AIDS, Hepatitis, HPV infection; pain; mental disorders;and inflammatory diseases.

As noted above, pharmaceutical compositions described herein are alsouseful in the prevention or treatment of viral infections, pain,inflammatory diseases, autoimmune diseases, and the like in a mammal.

In one aspect, the disclosure provides a method of reducing, retardingor stopping an abnormal cell growth comprising contacting the abnormalcell with a conjugate described herein, e.g., a compound of formula (I),FIG. 1), and/or formula (III) in an amount sufficient to retard, reduceor stop the abnormal cell growth, and wherein the abnormal cell growthis retarded, reduced or stopped.

In one aspect, the disclosure provides a method of killing a cell,comprising contacting the cell with a conjugate described herein, e.g.,a compound of formula (I), FIG. 1), and/or formula (III) in an amountsufficient to kill the cell, and wherein the cell is killed.

In one embodiment, the disclosure provides a method of killing a cell,comprising contacting the cell with a conjugate described herein, e.g.,a compound of formula (I), FIG. 1), and/or formula (III) in an amountsufficient to kill the cell, and wherein the cell is killed and furtherwherein the cell is a tumor cell.

In one aspect, the disclosure provides a method of treatment of amedical disorder in an individual suffering from the medical disorder,comprising administering to the individual an effective amount of acomposition comprising a conjugate described herein, e.g., a compound offormula (I), FIG. 1), and/or formula (III).

In one embodiment, the disclosure provides a method of treatment of amedical disorder in an individual suffering from the medical disordercomprising administering to the individual an effective amount of acomposition comprising a conjugate described herein, e.g., a compound offormula (I), FIG. 1), and/or formula (III) and further comprisingadministering sequentially or consecutively an additional therapy.

In one embodiment, the disclosure provides methods, wherein additionaltherapy is radiation therapy, chemotherapy, surgery, or combinationsthereof.

In one embodiment, the disclosure provides a method of treatment of amedical disorder in an individual suffering from the medical disordercomprising administering to the individual an effective amount of acomposition comprising a conjugated described herein, e.g., a compoundof formula (I), FIG. 1), and/or formula (III) and further comprisingadministering sequentially or consecutively an additional therapy andadministering at least one additional therapeutic agent.

In an aspect, the medical disorder treated is selected from tumors,cancers, infectious diseases, neurodegenerative diseases, bonedisorders, and cardiovascular diseases.

Finally, embodiments herein may include mixtures of the conjugatesdescribed herein, e.g., the compounds as represented by formula (I),FIG. 1), and/or formula (III).

While the disclosure has been particularly shown and described withreference to a number of embodiments, it would be understood by thoseskilled in the art that changes in the form and details may be made tothe various embodiments disclosed herein without departing from thespirit and scope of the disclosure and that the various embodimentsdisclosed herein are not intended to act as limitations on the scope ofthe claims.

V. EXAMPLES

Experimental Details

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the disclosure.

Proton NMR spectra were acquired on a Varian Inova 300 MHz or 500 MHzinstrument, while mass spectra were collected on one of two Agilent LCMSinstruments using electrospray ionization, either an 1100 series LC/MSDwith a ion trap analyzer or a 1200 series LC/MSD with a singlequadrupole analyzer. Conjugate mass spectra were acquired as detailed inthe example. All starting materials were purchased commercially and usedwithout purification, while solvents were purchased commercially anddried where necessary via methods well known in the art. The followingis a list of the abbreviations used in the Examples, with their fullchemical names in parentheses: Boc (N-tert-butoxycarbonyl), DCM(dichloromethane), DIEA (N,N-diisopropylethylamine), DMF(N,N-dimethylformamide), EtOAc (ethyl acetate), HATU(1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate), HCl (hydrochloric acid), HOAc (glacialacetic acid), HOAT (1-hydroxy-7-azabenzotriazole), HPLC(high-performance liquid chromatography), LCMS (tandem HPLC and massspectrometry), MeCN (acetonitrile), MeMgI (methyl magnesium iodide),MMAF (monomethylauristatin F), NaHCO₃ (sodium bicarbonate), Na₂SO₄(anhydrous sodium sulfate), NH₄Cl (ammonium chloride), t-BuOK (potassiumtert-butoxide), TFA (trifluoroacetic acid), THF (tetrahydrofuran).

Example 1:

The present Example illustrates the utility of conjugating a molecule toan antibody by bridging (i.e., rejoining) one or more disulfide bonds ofthe antibody with a linker compound of the present disclosure. Inparticular, a proof-of-concept linker molecule (i.e.,2-(bromo-methyl)acrylate (1) [Sigma Aldrich]), representative of thelinker-drug molecules of the present disclosure, was conjugated to anantibody at cysteine residues on the antibody, and the resultingconjugate molecule was assessed for stability using SDS-PAGE underreducing conditions.

Conjugate Preparation and Characterization

Briefly, a test monoclonal antibody of Fc isotype IgG1 (“mAbl,” 10mg/ml) in 50 mM HEPES, 150 mM NaCl, pH 7.5, was reduced with 1 mMdithiothreitol (0.006 mg per mg of antibody) at 37° C. for 30 min. Aftergel filtration (G-25, pH 4.5 sodium acetate), the methyl2-(bromo-methyl)acrylate (1, 1.2 equivalents/disulfide) in DMSO (10mg/ml) was added to the reduced antibody, the mixture was adjusted to pH7.0 with 1 M HEPES (pH 7.4), and allowed to react for 4 h. Immediatelyfollowing the alkylation, the antibody was oxidized with 0.5 mMdehydroascorbic acid (dhAA) for 4 h. The conjugate was purified by sizeexclusion chromatography. Protein concentrations were determined by UVspectral analysis. Size-exclusion HPLC established that all conjugateswere >95% monomeric. Conjugates were analyzed by reduced and non-reducedSDS-PAGE (FIG. 2).

Lane 1 in FIG. 2 shows molecular weight markers (KDaltons). Lane 2 inFIG. 2 is an intact mAbl (unconjugated, non-reduced). Lane 3 in FIG. 2is mAb1-1 conjugate treated with dhAA (conjugated, oxidized,non-reduced). The minor band in Lane 3 around 75 KDa is believed torepresent heavy-light chain “half-antibody” molecules (represented inFIG. 2 as “HL”). Lanes 4 and 5 in FIG. 2 are blank. Lane 6 in FIG. 2 ismAbl under reducing condition, which manifests as separate heavy chainand light chain bands. Lane 7 in FIG. 2 is mAb1-1 conjugate treated withdhAA under reducing condition. Lanes 8, 9, 10, 11, and 12 are blank. Asshown in FIG. 2, Lane 7, the mAb1-1 conjugate remained substantiallyintact under reducing conditions, suggesting that the disulfide linkagesformed by the bromomethacrylate compound served to stabilize theantibody and prevent dissociation of the immunoglobulin heavy and lightchains under reducing conditions.

Without being bound by any theory, the results of these experiments asanalyzed by reduced and non-reduced SDS-PAGE suggests that mAbl forms astable conjugate after the free sulfhydryl groups obtained via reducingthe disulfide bonds between the cysteine residues on mAb1 reacts withcompound 1. Further, the compound 1 tethers the two free sulfhydrylgroups on mAbl via covalent bonds. Therefore, mAb1-1 conjugate maintainsits structure under reducing conditions.

This proof-of-concept experiment therefore demonstrates that thelinker-drug compounds of the present disclosure, which are analogous tothe compound 1 test compound used in this Example, can be used toconjugate a variety of drugs, toxins and other molecules to antibodiesvia cysteine residues, while maintaining the structural integrity of theresulting antibody-drug conjugate.

Example 2:

Other proof-of-concept linker molecules (i.e., methyl3-bromo-2-(bromomethyl)propanoate (2) [Sigma Aldrich]), representativeof the linker-drug molecules of the present disclosure, were conjugatedto a reduced IgG1 antibody at cysteine residues on the antibody, and theresulting conjugate molecule was assessed using SDS-PAGE undernon-reducing and reducing conditions as in Example 1 (Tris/Glycine4-12%, Coomassie Stain).

2 -Bromomethyl-N-(1 -phenyl-ethyl)-acrylamide (6)

2-Bromomethyl-acrylic acid (2.0 g; 12.12 mmol) was dissolved in thionylchloride (2.6 mL; 4.3 g; 36.36 mmol) in a 50 mL 3-neck round bottomflask equipped with a magnetic stirrer, thermocouple, condenser andnitrogen inlet. This clear brown solution was refluxed at 90-92° C. for3 hours and then concentrated in vacuo to afford 2.01 grams (90.5%yield) of the corresponding acid chloride 4.

To another 50 mL 3-neck round bottom flask equipped with a magneticstirrer, addition funnel, thermocouple and nitrogen inlet was chargedR-1-phenyl-ethylamine (275 mg; 2.27 mmol), cat. DMAP (28 mg; 0.23 mmol,0.1 eq) and DCM (10 mL). This solution was chilled to 0° C. via an icebath. The 2-Bromomethyl-acryloyl chloride 4 (500 mg; 2.73 mmol, 1.2 eq)was also diluted with DCM (10 mL), chilled to 0° C. and slowly added tothe reaction mixture via addition funnel. The reaction was stirred inthe ice bath and slowly warmed to room temperature overnight. Thereaction was diluted with ethyl acetate and washed with Na₂SO₄, filteredand concentrated in vacuo to dryness, the crude product was dissolved inminimum amount of DCM and purified on silica gel (0-100% EtOAc inhexane). The cleanest fractions (by LC) were combined and concentratedto dryness giving the title compound as white solid (0.12 g, 20%).¹H-NMR (300 MHz, CDCl₃): 7.39-7.25 (m, 5H), 6.27 (br s, 1H), 5.84 (s,1H), 5.68 (s, 1H), 5.24-5.13 (m, 1H), 4.33-4.31 (app. d, 2H), 1.56 -1.54 (d, 3H)

3-Bromo-2-bromomethyl-N-(1-phenyl-ethyl)-propionamide (9)

3-Bromo-2-bromomethyl-propionic acid (1.0 g; 4.07 mmol) was dissolved inthionyl chloride (0.9 mL; 1.45 g; 12.2 mmol) in a 25 mL 3-neck roundbottom flask equipped with a magnetic stirrer, thermocouple, condenserand nitrogen inlet. This clear brown solution was refluxed at 90-92° C.for 3 hours and then concentrated in vacuo to afford 0.89 grams (83%yield) of the corresponding acid chloride. ¹H-NMR (300 MHz, CDCl₃): δ3.85-3.75 (m, 4H), 3.60 (pentet, 1H, J=9 Hz).

To another 50 mL 3-neck round bottom flask equipped with a magneticstirrer, addition funnel, thermocouple and nitrogen inlet was chargedR-1-phenyl-ethylamine (149 mg; 1.23 mmol), cat. DMAP (18 mg; 0.15 mmol,0.1 eq) and DCM (10 mL). This solution was chilled to 0 ° C. via an icebath. The 3-Bromo-2-bromomethyl-propionyl chloride 8 (390 mg; 1.48 mmol,1.2 eq) was also diluted with DCM (10 mL), chilled to 0° C. and slowlyadded to the reaction mixture via addition funnel. The reaction wasstirred in the ice bath and slowly warmed to room temperature overnight.The reaction was diluted with ethyl acetate and washed with Na₂SO₄,filtered and concentrated in vacuo to dryness, the crude product wasdissolved in minimum amount of DCM and purified on silica gel (0-100%EtOAc in hexane). The cleanest fractions (by LC) were combined andconcentrated to dryness giving the title compound as white solid (0.13g, 24%). ¹H-NMR (300 MHz, CDCl₃): 7.36-7.25 (m, 5H), 6.03-6.01 (d, 1H),5.22-5.13 (m, 1H), 3.65 - 3.45 (m, 4H), 2.92-2.82 (m, 1H), 1.55-1.53 (d,3H)

Conjugate Preparation and Characterization

Briefly, a test monoclonal antibody of Fc isotype IgG1 (“mAbl,” 10mg/ml) in 50 mM HEPES, 150 mM NaCl, pH 7.5, was reduced with 1 mMdithiothreitol (0.006 mg per mg of antibody) at 37° C. for 30 min. Aftergel filtration (G-25, pH 4.5 sodium acetate), the reunion reagent 1, 2,6, or 9 (1.2 equivalents/disulfide) in DMSO (10 mg/ml) was added to thereduced antibody, the mixture was adjusted to pH 7.0 with 1 M HEPES (pH7.4), and allowed to react for 4 h. Immediately following thealkylation, the antibody was oxidized with 0.5 mM dehydroascorbic acid(dhAA) for 4 h. The conjugate was purified by size exclusionchromatography. Protein concentrations were determined by UV spectralanalysis. Size-exclusion HPLC established that all conjugates were >95%monomeric. FIG. 3 contains the reduced and non-reduced SDS-PAGE gel ofthe conjugates (Tris/Glycine 4-12%, Coomassie Stain).

Lane 1 in FIG. 3 shows molecular weight markers (KDaltons). Lane 2 inFIG. 3 is an intact mAb1 (unconjugated, non-reduced). Lanes 3 and 4 aremAb1-6 and mAb1-9 conjugates, respectively. The major band in Lanes 3and 4 around 75 KDa corresponds to the heavy-light chain “half-antibody”molecules (represented in FIG. 3 as “HL”). Lanes 5 and 6 in FIG. 3 aremAb1-2 and mAb1-1 conjugates, respectively. The major band around 150KDa for both conjugates corresponds to the intact antibody. Lane 7 is ablank. Lane 8 in FIG. 3 is mAb1 under reducing conditions, whichmanifests as separate heavy chain and light chain bands. Lanes 9 and 10are mAb1-6 and mAb1-9 conjugates, respectively, under reducingconditions. In those lanes, 3 bands are present that correspond to lightchain (25 KDa), heavy chain (50 KDa), and heavy-light chain or“half-antibody” molecules. Lanes 11 and 12 in FIG. 3 are mAb1-2 andmAb1-1 conjugates, respectively, under reducing conditions. The majorband around 150 KDa for both conjugates corresponds to the intactantibody. As shown in FIG. 3, Lanes 11 and 12, the mAb1-2 and mAb1-1conjugates remained substantially intact under reducing conditions,suggesting that the disulfide linkages formed by the disulfide reunioncompounds served to stabilize the antibody and prevent dissociation ofthe immunoglobulin heavy and light chains under reducing conditions.

Without being bound by any theory, the results of these experiments asanalyzed by reduced and non-reduced SDS-PAGE suggests that mAbl forms astable conjugate after the free sulfhydryl groups obtained via reducingthe disulfide bonds between the cysteine residues on mAbl reacts withthe disulfide reunion compounds. Further, the tethers formed on the twofree sulfhydryl groups on mAb1 are covalent bonds. Therefore, mAb1-6 andmAb1-9 conjugates maintain a majority of their structure under reducingconditions and in the mAb1-2 and mAb1-1 conjugates approximately all ofthe IgG structure remains intact.

This additional proof-of-concept experiment therefore demonstrates thatthe linker-drug compounds of the present disclosure, which are analogousto compounds 1, 2, 6, and 9 used in this Example, can be used toconjugate a variety of drugs, toxins and other molecules to antibodiesvia cysteine residues, while maintaining the structural integrity of theresulting antibody-drug conjugate.

Example 3

Linker Synthesis

-(3-Bromo-2-bromomethyl-propionylamino)-hexanoic acid (13) Step 1:Preparation of 6-Amino-hexanoic acid tert-butyl ester (11)

To a 50 mL round bottom flask equipped with a magnetic stirrer andnitrogen inlet was charged 6-aminohexanoic acid (2.0 g; 15 mmol) andthionyl chloride (5.0 mL; 69 mmol; 4.5 equiv.). This solution wasstirred at or below 30° C. for 2 hours and concentrated in vacuo todryness. To the tan semi-solid a slurry of sodium bicarbonate (2.6 g; 30mmol; 2.0 equiv.) in t-BuOH (5.0 mL; 87 mmol; 5.7 equiv.) was added andthe slurry was stirred at ambient temperature for another 2 h. Thebutanol was removed in vacuo at 40° C. The thick white slurry wasdiluted with ethyl acetate and washed with 4 portions of 1 N NaOH, 3portions of H₂O, 1 portion of brine. The organics were dried overNa₂SO₄, filtered and concentrated to afford 2.2 g (77% yield) as acolorless oil. MS (ESI, pos.): calc'd for C₁₀H₂₁NO₂, 187.3; found 188.4(M+H), ¹H-NMR (300 MHz, CDCl₃): δ 2.68-2.64 (m, 2H), 2.21 - 2.16 (m,2H), 1.62 - 1.52 (m, 2H), 1.48-1.38 (m, 9H), 1.36-1.20 (m, 2H), 1.09 (m,2H).

Step 2: Preparation of 6-(3-Bromo-2-bromomethyl-propionylamino)-hexanoicacid tert-butyl ester (12)

To a 50 mL round bottom flask equipped with a magnetic stirrer andnitrogen inlet was charged 6-aminohexanoic acid tert-butyl ester 11(0.50 g; 2.7 mmol) and dimethylaminopyridine (0.03 g; 0.27 mmol; 0.10equiv.) in DCM (5.0 mL). This solution was chilled to 0° C. via an icebath. 3-Bromo-2-bromomethyl-propionyl chloride 8 (0.90 g; 3.4 mmol; 1.2equiv.) was dissolved in DCM (5 mL) and slowly added to the reactionmixture at 0° C. Stir and slowly warm to ambient temperature overnight.Dilute reaction mixture with ethyl acetate, wash the organic mixturewith H₂O, 5% NaHCO₃ and brine. The organics were dried over Na₂SO₄,filtered, concentrated and purified on a silica gel column eluting withO-100% ethyl acetate in hexanes to afford 0.49 g (42% yield) as a clearyellow oil. ¹H-NMR (300 MHz, CDCl₃): δ 5.92 (br s, 1H), 3.64-3.58 (m,2H), 3.54-3.48 (m, 2H), 3.36-3.29 (m, 2H), 2.89-2.83 (m, 1H), 2.24-2.20(m, 2H), 1.65-1.51 (m, 4H), 1.44-1.35 (m, 11H).

Step 3: Preparation of 6-(3-Bromo-2-bromomethyl-propionylamino)-hexanoicacid (13)

To a 50 mL round bottom flask equipped with a magnetic stirrer andnitrogen inlet was charged6-(3-Bromo-2-bromomethyl-propionylamino)-hexanoic acid tert-butyl ester12 (0.26 g; 0.62 mmol) and trifluoroacetic acid (0.70 mL; 9.3 mmol; 15equiv.) in DCM (10 mL). This solution was stirred at ambient temperatureovernight, concentrated to dryness, dissolved in acetonitrile and H₂O(1.0 mL each), frozen and lyophilized to afford 0.22 g (100%) as asolid. MS (ESI, pos.): calc'd for C₁₀H₁₇Br₂NO₃, 359.0 found 358.0,360.0, 362.0 (M+H), 380.0, 382.0, 384.0 (M+Na), 356.0, 358.0, 360.0(M−H). ¹H-NMR (300 MHz, CDCl₃): δ 11.97 (s, 1H), 8.20-8.16 (m, 1H),3.58-3.56 (d, 4H), 3.11-3.04 (m, 2H), 3.02-2.97 (m, 1H), 2.21-2.16 (m,2H), 1.54-1.37 (m, 4H), 1.33-1.29 (m, 2H).

6-(3-Bromo-2-bromomethyl-propionyloxy)-hexanoic acid (17) Step 1:Preparation of 6-Hydroxy-hexanoic acid tert-butyl ester (15)

To a 250 mL round bottom flask equipped with a magnetic stirrer,thermocouple, condenser and nitrogen inlet was charged 6-hexanolactone14 (3.7 g; 32 mmol) and t-BuOH (100 mL) in DCM followed by t-BuOK (3.9g; 35 mmol; 1.1 equiv.). This solution was stirred and heated to refluxfor 3 hours. The solution was diluted with toluene, washed with waterand brine, dried over Na₂SO₄, filtered and concentrated to afford 4.8 g(80% yield) as a clear yellow oil. ¹H-NMR (500 MHz, CDCl₃): δ 3.65-3.62(m, 2H), 2.32-2.29 (m, 1H), 2.23-2.20 (m, 2H), 1.66-1.55 (m, 4H), 1.43(s, 9H), 1.41-1.35 (m, 2H).

Step 2: Preparation of 6-(3-Bromo-2-bromomethyl-propionyloxy)-hexanoicacid tert-butyl ester (16)

To a 50 mL round bottom flask equipped with a magnetic stirrer andnitrogen inlet was charged 6-Hydroxy-hexanoic acid tert-butyl ester 15(0.50 g; 2.7 mmol) and dimethylaminopyridine (0.03 g; 0.27 mmol; 0.10equiv.) in DCM (5.0 mL). This solution was chilled to 0° C. via an icebath. 3-Bromo-2-bromomethyl-propionyl chloride 8 (0.90 g; 3.4 mmol; 1.3equiv.) was dissolved in DCM (5.0 mL) and slowly added to the reactionmixture at 0° C. The mixture was stirred and slowly warmed to ambienttemperature overnight. The reaction mixture was diluted with ethylacetate, wash the organic mixture with H₂O, 5% NaHCO₃ and brine. Theorganics were dried over Na₂SO₄, filtered, concentrated and purified ona silica gel column eluting with O-100% ethyl acetate in hexanes toafford 0.75 g (78% yield) as a clear yellow oil. ¹H-NMR (300 MHz,CDCl₃): δ 4.21-4.15 (m, 2H), 3.80-3.68 (m, 4H), 3.21-3.15 (m, 1H),2.40-2.31 (m, 2H), 1.71-1.59 (m, 4H), 1.42-1.37 (m, 11H).

Step 3: Preparation of 6-(3-Bromo-2-bromomethyl-propionyloxy)-hexanoicacid (17)

To a 50 mL round bottom flask equipped with a magnetic stirrer andnitrogen inlet was charged6-(3-Bromo-2-bromomethyl-propionyloxy)-hexanoic acid tert-butyl ester 16(0.70 g; 1.7 mmol) and trifluoroacetic acid (1.3 mL; 17 mmol; 10 equiv.)in DCM (10 mL). This solution was stirred at ambient temperatureovernight, concentrated and purified on a silica gel column eluting with0-100% ethyl acetate in hexanes to afford 0.34 g (56% yield) as a clearcolorless oil. MS (ESI, pos.): calc'd for C₁₀H₁₆Br₂O₄, 360.0; found380.8, 382.8, 384.8 (M+Na), 276.0, 278.0 (M−H). ¹H-NMR (300 MHz, CDCl₃):δ 4.21-4.16 (m, 2H), 3.80-3.67 (m, 4H), 3.22-3.16 (m, 1H), 2.40-2.35 (m,2H), 1.75-1.63 (m, 4H), 1.50-1.39 (m, 2H).

6-((3-bromo-2-(bromomethyl)propanoyl)oxy)-6-methylheptanoic acid (22)Step 1: Preparation of 6-Hydroxy-6-methyl-heptanoic acid (19)

To a 250 mL 3-neck round bottom flask equipped with a magnetic stirrer,addition funnel, condenser, thermocouple and nitrogen inlet was charged5-acetylvaleric acid 18 (1.0 g; 6.9 mmol) and anhydrous THF (50 mL).This solution was stirred and chilled to −78° C. via dry ice/acetonebath. To this cold solution 1.4 M MeMgI in THF/toluene (29 mL; 40 mmol;5.8 eq) was added slowly via addition funnel over 15 min. The reactionwas stirred at −78° C. for 2 h, then slowly warmed to −10° C. andsaturated aq. NH₄Cl solution was added to quench the excess Grignard.The biphasic mixture was warmed to ambient temperature, diluted withethyl acetate, the organic layer was discarded. The aqueous layer wasacidified with 1 M HCl and extracted with ethyl acetate (3×50 mL). Theorganic phases were combined, washed with brine, dried over Na₂SO₄,filtered and concentrated to afford 0.63 g (57% yield) as a clearcolorless oil. ¹H-NMR (500 MHz, CDCl₃): δ 2.39-2.36 (m, 2H), 1.68-1.62(m, 2H), 1.51-1.47 (m, 2H), 1.45-1.40 (m, 2H), 1.21 (s, 6H).

Step 2: 6-Hydroxy-6-methyl-heptanoic acid tert-butyl ester (20)

To a 100 mL 3-neck round bottom flask equipped with a magnetic stirrer,addition funnel, condenser, thermocouple and nitrogen inlet was charged6-Hydroxy-6-methyl-heptanoic acid 19 (0.68 g; 4.2 mmol) and DCM (30 mL).This solution was stirred at ambient temperature and theN,N′-diisopropyl-O-tert-butyl isourea (2.6 g; 13 mmol) was addeddropwise via addition funnel. The reaction was stirred overnight andthen heated to 40° C. for 3 h. The solvent was removed and the crudeproduct was purified on a silica gel flash column eluting with 0-50%ethyl acetate in hexanes, and product fractions evaporated in vacuogiving the title compound as a clear colorless oil (0.28 g, 30% yield).¹H-NMR (500 MHz, CDCl₃): δ 2.25-2.21 (t, 2H), 1.63-1.57 (q, 2H),1.49-1.46 (m, 2H), 1.44 (m, 9H), 1.41-1.36 (m, 2H), 1.21 (s, 6H).

Steps 3-4: Preparation of compounds 21 and 22

Compound 20 can be acylated with compound 8 using DMAP to yield compound21 and trifluoroacidic acid removal of the t-butyl protecting groupwould furnish the desired hindered functionalized acid linker 22 thatcan be couple to payloads and linker payloads.

Example 4

Maytansinoid Linker Payloads

Compound 23 was prepared as a white solid (0.074 g, 63%) according tothe method described in WO2014145090. MS (ESI, pos.): calc'd forC₅₅H₇₈ClN₉O₁₅, 1139.5; found 1141.4 (M+H).

6-(3-Bromo-2-bromomethyl-propanamido)-caproamido-L-valine-L-citrulline-p-amino-benzylcarbamoyl-N-methyl-beta-alanine-N-methyl-L-alanine-maytansine-3-ester (24)

Compound 23 (0.016 g, 0.013 mmol), compound 13 (0.016 g, 0.045 mmol) andHATU (0.024 g, 0.063 mmol) were weighed into a dry round-bottom flask,dissolved in DMF (3.0 mL), and treated with DIEA (0.010 mL, 0.057 mmol)via syringe. The flask was purged with argon, sealed with a rubberseptum, and stirred at ambient temperature for 22 h. The reaction wasdiluted with water and purified on a 100 g C18 Aq RediSep Gold columnvia ISCO CombiFlash system (20-80% MeCN in water, 0.05% HOAc both, over15 mins, 50 mL/min), and the cleanest fractions frozen and lyophilizedgiving the title compound as a white solid (0.015 g, 75%). MS (ESI,pos.): calc'd for C₆₅H₉₃Br₂ClN₁₀O₁₇, 1480.5/1481.5 (most abundantisotopes); found 1481.5/1483.6 (M+H).

2-(Bromomethyl)-acrylamido-6-caproamido-L-valine-L-citrulline-p-aminobenzylcarbamoyl-N-methyl-beta-alanine-N-methyl-L-alanine-maytansine-3-ester (25)

Compound 24 (0.021 g, 0.014 mmol) was dissolved in MeCN (4.0 mL),treated with pH 9.00 buffer (4.0 mL, Fisher Chemical #SB114-500), theflask purged with argon, sealed with a rubber septum, and stirred atambient temperature for 20 h. The reaction was frozen on dry ice,lyophilized to a solid, dissolved in 1:1 MeCN/water, and purified on a50g C18 Aq RediSep Gold column via ISCO CombiFlash system (20-80% MeCNin water, 0.05% HOAc both, over 12 mins, 40 mL/min), and the cleanestfractions frozen and lyophilized giving the title compound as a whitesolid (0.013 g, 65%). MS (ESI, pos.): calc'd for C₆₅H₉₂BrClN₁₀O₁₇,1400.6 (most abundant isotope); found 1401.5 (M+H).

2-(Bromomethyl)-acrylato-6-caproamido-L-valine-L-citrulline-p-aminobenzylcarbamoyl-N-methyl-beta-alanine-N-methyl-L-alanine-maytansine-3-ester (26)

Compound 17 (0.022 g, 0.061 mmol), compound 23 (0.024 g, 0.020 mmol),and HATU (0.038 g, 0.10 mmol) were weighed into a dry round-bottomflask, dissolved in DMF (3.0 mL), and treated with DIEA (0.02 mL, 0.12mmol) via syringe. The flask was purged with argon, sealed with a rubberseptum, and stirred at ambient temperature for 24 h. The reaction wasconcentrated in vacuo to an oil, dissolved in 1:1 MeCN/water, treatedwith 1 drop of 10% aq. HOAc, and purified on a 50g C18 Aq RediSep Goldcolumn via ISCO CombiFlash system (20-80% MeCN in water, 0.05% HOAcboth, over 12 mins, 40 mL/min), and the cleanest fractions frozen andlyophilized giving the title compound as a white solid (0.012 g, 43%).MS (ESI, pos.): calc'd for C₆₅H₉₁BrClN₉O₁₈, 1401.5 (most abundantisotope); found 1402.6 (M+H).

Example 5

Auristatin Linker Payloads

6-(3-Bromo-2-bromomethyl-propanamido)-caproamido-monomethylauristatin F(28)

Compound 13 (0.040 g, 0.11 mmol) and HATU (0.033 g, 0.087 mmol) wereweighed into a dry round-bottom flask, dissolved in DMF (2.0 mL), andtreated with DIEA (0.030 mL, 0.17 mmol) via syringe. The flask waspurged with argon, sealed with a rubber septum, and stirred at ambienttemperature for 0.5 h. A solution of Monomethyl Auristatin F 27 (0.034g, 0.046 mmol) in DMF (1.0 mL) was then added, the flask purged againwith argon and resealed, and the reaction stirred another 18 h. Thereaction was diluted with water (3 mL) and purified on a 100 g C18 AqRediSep Gold column via ISCO CombiFlash system (20-80% MeCN in water,0.05% HOAc both, over 17 mins, 50 mL/min). The product-containingfractions by LCMS were combined, frozen in a dry ice/acetone bath, andlyophilized giving the title compound as a white solid (0.024 g, 44%).MS (ESI, pos.): calc'd for C₄₉H₈₀Br₂N₆O₁₀, 1072.4 (most abundantisotope); found 1073.5 (M+H).

2-(Bromomethyl)-acrylamido-6-caproamido-monomethylauristatin F (29)

Compound 28 (0.013 g, 0.012 mmol) was dissolved in MeCN (2.0 mL),treated with pH 9.00 buffer (2.0 mL, Fisher Chemical #SB114-500), theflask purged with argon, sealed with a rubber septum, and stirred atambient temperature for 40 h. The reaction showed only 56% conversion toproduct by LCMS, so saturated aq. NaHCO3 was added to bring the pH up to8.5 and the reaction stirred another 7 h at ambient temperature. Thereaction was acidified with 10% aq. HOAc (1.0 mL) and purified on a 50 gC18 Aq RediSep Gold column via ISCO CombiFlash system (20-80% MeCN inwater, 0.05% HOAc both, over 12 mins, 40 mL/min). The product-containingfractions by LCMS were combined, frozen in a dry ice/acetone bath, andlyophilized giving the title compound as a white solid (9 mg). This wasonly 85% pure by HPLC, so it was repurified on a 30 g C18 Aq RediSepGold column via ISCO CombiFlash system (20-80% MeCN in water, 0.05% HOAcboth, over 12 mins, 30 mL/min), and the cleanest fractions frozen andlyophilized giving the title compound as a white solid (0.005 g, 42%).MS (ESI, pos.): calc'd for C₅₅H₇₈ClN₉O₁₅, 990.5/992.5 (most abundantisotopes); found 991.5/993.5 (M+H).

Conjugate Preparation and Characterization

An anti-PRLR monoclonal antibody from US20150056222 (WO2015026907),H1H6765P, and a non-targeting monoclonal antibody both with Fc isotypeIgG1 (10 mg/ml) in 50 mM HEPES, 150 mM NaCl, pH 7.5, was reduced with 1mM dithiothreitol (0.006 mg per mg of antibody) at 37° C. for 30 min.After gel filtration (G-25, pH 4.5 sodium acetate), one of the compounds26, 28, or 29 (1.2 equivalents/disulfide) in DMSO (10 mg/ml) was addedto the reduced antibody, the mixture was adjusted to pH 7.0 with 1 MHEPES (pH 7.4), and allowed to react for 4 h. Immediately following thealkylation, the antibody was oxidized with 0.5 mM dehydroascorbic acid(dhAA) for 4 h or allowed to air oxidized. The conjugate was purified bysize exclusion chromatography. Protein concentrations were determined byUV spectral analysis. Size-exclusion HPLC established that allconjugates were >95% monomeric. Drug to antibody ratios (DAR) for themaytansinoid conjugates of 26 were determined using the extinctioncoefficients 280 nm ═8115 cm⁻¹ M⁻¹ and 252 nm=49407 cm⁻¹ M⁻¹, whichyielded a DAR of 2. The auristatin conjugate DARs were not determined.Conjugates were analyzed by reduced and non-reduced SDS-PAGE and FIG. 4contains the gel (Tris/Glycine 4-12%, Coomassie Stain).

Lane 1 in FIG. 4 shows molecular weight markers (KDaltons). Lane 2 inFIG. 4 contains intact H1H6765P antibody (unconjugated, non-reduced). Inlanes 3 and 4 are H1H6765P-28 and H1H6765P-29 conjugates, respectively.Three major bands are present in Lanes 3 and 4 around 75, 125, and 150KDa corresponding to the heavy-light chain “half-antibody,”heavy-heavy-light antibody, and intact antibody conjugates. In Lane 5 ofFIG. 4 is H1H6765P-26 with only major bands around 75 and 150 KDacorresponding to the heavy-light chain “half-antibody,” and intactantibody conjugates respectively. Lanes 6 and 7 are blank. Lane 8 inFIG. 4 contains H1H6765P under reducing conditions, which manifests asseparate heavy chain and light chain bands. Lanes 9 and 10 areH1H6765P-28 and H1H6765P-29 conjugates, respectively, under reducingconditions. In those lanes, 3 major bands around 25, 50, and 75 KDacorrespond to the light chain, heavy chain, and heavy-light chain“half-antibody” conjugates, respectively. Lane 11 in FIG. 4 containsconjugate H1H6765P-26 under reducing conditions. In that lane, the bandspresent around 75, 125, and 150 KDa correspond to the heavy-light chain“half-antibody,” heavy-heavy-light antibody, and intact antibodyconjugates. As shown in FIG. 4, Lanes 9-10 the conjugates remainedpartially intact under reducing conditions. In lane 11 the H1H6765P-26remained substantially intact suggesting that the disulfide linkagesformed by the disulfide reunion linker payload served to stabilize theantibody and prevent dissociation of the immunoglobulin heavy to lightand heavy to heavy chains under reducing conditions.

Conjugate Analysis by Mass Spectrometry

Conjugates H1H6765P-28 and H1H6765-26 were analyzed by ESI-MS on aThermo Q-Exactive (Hybrid Quadrupole-Orbitrap) equipped with a ShimadzuHPLC with prominence LC-20AD pump, SIL-20AC HT autosample and SPD-20A UVdetector.

Each conjugate was diluted with MilliQ water to 1 mg/mL. PNGase F(P0704L, New England Biolabs) was diluted with MilliQ water to75,000U/mL. 5 uL of the diluted PNGase F was added to 50 uL of eachsample and the samples were incubated at 37° C. with shaking (500 RPM)overnight. 5 uL of each sample was injected to LC-MS system.

Chromatographic separation was achieved by a LC gradient composing ofmobile phase A (0.1% formic acid in water) and mobile phase B (0.1%formic acid in acetonitrile) with the gradient shown in Table 1. Flowrate was set at 0.4 ml/min. Waters BEH C18 column (50X2.1 mm 1.7umparticle size) was kept at 60° C. during the analysis. The flow between3-14 min was directed to MS and others were directed to waste.

TABLE 1 LC gradient Minute 0.01 3 13.9 14 16 17 24 % B 10 10 90 95 95 1010

Mass Spectrometric Setting

Thermo Q-Exactive with HESI ionization source was used to detect eluentsfrom LC. The other major parameters are listed in Table 2.

TABLE 2 Q-Exactive parameters for intact mass analysis In Parameter ScanRange Resolution AGC Maximum IT source CID Value 700-4000 Da 17,500 5e6200 ms 80.0 eV Parameter Spray Voltage Aug gas Spare gas Capillarytemperature Value 4400 V 10 8 250° C.

Collected mass spectrum was deconvoluted by using Thermo ProteinDeconvolution software. Manual ReSpect™ was selected as the ExperimentTypes. Noise rejection was set to 95% confidence and mass tolerance wasset to 25 ppm.

FIG. 5 contains the mass spectrum of the deglycosylated conjugate ofH1H6765-28. The mass peaks are consistent with either an intact,heavy-heavy-light, or “half-antibody” by disulfide reunion provided bylinker payload 28 or oxidation. The degree in which 28 has made covalentbonds between the sulfhydryls manifests as discreet mass increasescorresponding to reacted linker-payload (993 Da, -1 Bromine or 913 Da,-2 Bromines). The mass spectrum is summarized in Table 3.

TABLE 3 Mass Difference from Peak adjacent peak of the same Matched (inFIG. 5) Mass (Da) species (Da) Components* P1 74276.797 HL P2 75188.813912.0 HL + 1 drug P3 122479.523 HHL P4 123390.578 911.1 HHL + 1 drug P5124303.344 912.8 HHL + 2 drugs P6 144906.469 HHLL P7 145819.813 913.3HHLL + 1 drug P8 146731.172 911.4 HHLL + 2 drugs P9 147643.922 912.8HHLL + 3 drugs *Note: H refers to heavy chain of mAb; L for light chainof mAb; HL for half mAb; HHL refers to two heavy chains with one lightchain; HHLL refers to the full mAb. The increase of mass from additionof one drug is 913 Da.

FIG. 6 contains the mass spectrum of the deglycosylated conjugate ofH1H6765-26. The mass peaks are consistent with either an intact or“half-antibody” by disulfide reunion provided by linker payload 26 oroxidation. The degree in which 26 has made covalent bonds between thesulfhydryls manifests as discreet mass increases corresponding toreacted linker-payload (1323 Da). The mass spectrum is summarized inTable 4.

TABLE 4 Mass Peak (in Difference from adjacent peak Matched FIG. 6) Mass(Da) of the same species (Da) Components* P1 72652.508 HL P2 73974.6091122.2 HL + 1 drug P3 75181.383 1206.8 HL + 2 drugs P4 76416.953 1235.6HL + 3 drugs P5 145105.625 HHLL P6 146425.141 1319.5 HHLL + 1 drug P7147749.703 1324.6 HHLL + 2 drug P8 149077.906 1328.2 HHLL + 3 drug P9150483.453 1405.5 HHLL + 4 drug *Note: H refers to heavy chain of mAb; Lfor light chain of mAb; HL for half mAb; HHL refers to two heavy chainswith one light chain; HHLL refers to the full mAb. Due to theheterogeneity of each species, the increase of mass from addition of onedrug may be different from the nominal 1322.9 Da

Example 6

In Vitro Cytotoxicity Assays

In this Example, the ability of various antibody-drug conjugates to killantigen-expressing tumor cells in vitro was assessed.

Cells were seeded in Nunclon Delta Surface 96 well plates at 1000(HEK293 and HEK293/PRLR) or 6000 (T47D) cells per well, and cells weregrown overnight in complete growth media. For cell viability curves,serially diluted conjugates or free representative payloads were addedto the cells at final concentrations ranging from 100 nM to 5 pM andincubated for 3 days. To measure viability, cells were incubated withCCK8 (Dojindo) for the final 1-3 hours and the absorbance at 450nm(OD450) was determined on a Victor X4 (PerkinElmer). Background OD450levels (CCK8) from digitonin (40 nM) treated cells were subtracted fromall wells and viability is expressed as a percentage of the untreatedcontrols. IC50 values were determined from a four-parameter logisticequation over a 10-point response curve (GraphPad Prism). Maytansinoidconjugate curves and IC50 values are corrected for payload equivalentsbased on the UV DAR value. For the auristatin (MMAF) conjugate, the IC50values are assigned a DAR of 1 (i.e., equal to antibody concentration)since a good estimate cannot be obtained by UV or mass spectrometry.

In FIG. 7, the maytansinoid payload (compound 7 from WO2014145090) wasnot active up to 100 nM in any of the cell lines assayed as expected.The isotype control cathepsin B cleavable conjugate, Isotype Control-26,was similarly inactive. However, the targeted cathepsin B cleavableH1H6765P-26 conjugate was active with an IC50 of ˜4 nM in the engineeredcell line HEK293/hPRLR and 0.52 nM in the endogenous cell line T47D.H1H6765P-26 was not active up to 100 nM in the native cell line HEK293(no target expressed) as expected. The unconjugated H1H6765P was devoidof activity in these assays.

In FIG. 8, compound 27 (MMAF) payload was not active up to 100 nM in anyof the cell lines assayed as expected. The control non-cleavableconjugates, Isotype Control-28 or Isotype Control-29, were similarlyinactive. However, the targeted non-cleavable conjugates, H1H6765P-28 orH1H6765P-29, were active with IC50 values of ˜0.04 and ˜0.03 nM,respectively, in the engineered cell line HEK293/hPRLR and 0.03 and 0.03nM, respectively, in the endogenous cell line T47D. Neither H1H6765P-28or Control-29 were active up to 100 nM in the native cell line HEK293(no target expressed) as expected. The unconjugated H1H6765P was devoidof activity in these assays.

1.-30. (canceled)
 31. An antibody-drug conjugate comprising an antibody,or antigen binding fragment thereof, wherein the antibody or antigenbinding fragment thereof is conjugated to at least one moiety of Formula(A):

wherein: Ab-S1l is a bond to a cysteine sulfur atom of the antibody orantigen binding fragment thereof; Ab-S2 is a bond to a cysteine sulfuratom of the antibody or antigen binding fragment thereof; X is—N(R^(A))— or —O—; wherein R^(A) is a hydrogen atom or alkyl; R^(N) andR^(M) are each, independently, a hydrogen atom or alkyl; A is absent ora spacer comprising a peptide, wherein the peptide comprises 2-20 aminoacids; D is a biologically active molecule; and b is an integer from 2to
 8. 32. The conjugate of claim 31, wherein the antibody or antigenbinding fragment thereof comprises at least one moiety of Formula (A)wherein Ab-S1 is a bond to a cysteine sulfur atom of a first heavy chainof the antibody or antigen binding fragment thereof and AB-S2 is a bondto a cysteine sulfur atom of a second heavy chain of the antibody orantigen binding fragment thereof
 33. The conjugate of claim 31, whereinthe antibody or antigen binding fragment thereof comprises: two moietiesof Formula (A) wherein Ab-S1 is a bond to a cysteine sulfur atom of afirst heavy chain of the antibody or antigen binding fragment thereofand AB-S2 is a bond to a cysteine sulfur atom of a second heavy chain ofthe antibody or antigen binding fragment thereof; and (ii) two moietiesof Formula (A) wherein Ab-S1 is a bond to a cysteine sulfur atom of alight chain of the antibody or antigen binding fragment thereof andAB-S2 is a bond to a cysteine sulfur atom of a heavy chain of theantibody of antigen binding fragment thereof.
 34. The conjugate of claim31, wherein the conjugate comprises an anti-PRLR antibody.
 35. Theconjugate of claim 31, wherein X is —NH—.
 36. The conjugate of claim 31,wherein X is —O—.
 37. The conjugate of claim 31, wherein X is —NH— or—O— and R^(N) and R^(M) are both hydrogen atoms.
 38. The conjugate ofclaim 37, wherein b is
 4. 39. The conjugate of claim 31 wherein A is:

wherein

is the bond to D, and AA₁ and AA₂ are each, independently, an aminoacid.
 40. The conjugate of claim 39, wherein A is:

wherein

is the bond to D.
 41. The conjugate of claim 31, wherein A is absent.42. The conjugate of claim 31, wherein D is an auristatin ormaytansinoid.
 43. The conjugate of claim 31, wherein D is MMAE, MMAD, orMMAF.
 44. The conjugate of claim 31, wherein D is a maytansinoid. 45.The conjugate of claim 31, wherein D is DM1 or DM4.
 46. The conjugate ofclaim 31, wherein D is:


47. The conjugate of claim 31, wherein D is:


48. The conjugate of claim 31, wherein the moiety of Formula (A) is:


49. The conjugate of claim 31, wherein the moiety of Formula (A) is:


50. The conjugate of claim 31, wherein the moiety of Formula (A) is:

wherein D is a maytansinoid.
 51. The conjugate of claim 31, wherein themoiety of Formula (A) is:

wherein D is a maytansinoid.
 52. The conjugate of claim 31, wherein themoiety of Formula (A) is:


53. The conjugate of claim 31, wherein the moiety of Formula (A) is:


54. The conjugate of claim 31, wherein the moiety of Formula (A) is:

wherein D is an auristatin.
 55. The conjugate of claim 31, wherein themoiety of Formula (A) is:

wherein D is an auristatin.
 56. The conjugate of claim 31, wherein themoiety of Formula (A) is:


57. A compound of Formula (L1):

wherein: R^(U) is

wherein LG¹ and LG², independently at each occurrence, are a leavinggroup; X is —N(R^(A))— or —O—; wherein R^(A) is a hydrogen atom oralkyl; R^(N) and R^(M) are each, independently, a hydrogen atom oralkyl; A is absent or a spacer comprising a peptide, wherein the peptidecomprises 2-20 amino acids; D is a biologically active molecule; and bis an integer from 2 to
 8. 58.-80. (canceled)
 81. A pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundas claimed in claim 31 or a pharmaceutically acceptable salt thereof andone or more pharmaceutically acceptable carriers, diluents, orexcipients.
 82. A method of reducing, retarding or stopping an abnormalcell growth comprising contacting the abnormal cell with a compound ofclaim 31, in an amount sufficient to retard, reduce or stop the abnormalcell growth, and wherein the abnormal cell growth is retarded, reducedor stopped.
 83. A method of killing a cell, comprising contacting thecell with a compound of claim 31, in an amount sufficient to kill thecell, and wherein the cell is killed.
 84. A method of treatment of amedical disorder in an individual suffering from the medical disorder,comprising administering to the individual an effective amount of acomposition comprising a compound of claim 31.